p53 mt cancer cells  (ATCC)

Journal: Nucleic Acids Research

Article Title: HIF-transcribed p53 chaperones HIF-1α

doi: 10.1093/nar/gkz766

Figure Lengend Snippet: HIF-1α correlates with p53 expression in hypoxic zones of human cancers. ( A ) Immunohistochemical staining for p53 and HIF-1α shows their distribution in ten different regions of pancreatic and kidney tumors. The HIF-1α expression shows great intra-tumoral heterogeneity within the hypoxic microenvironment of both cancers. Ten fields marked 1–10 with variable HIF-1α expression were selected, and p53 expression in such areas was observed. A zoomed view (40×) of each of these ten fields shows high-resolution staining for HIF-1α and p53, H&E staining is used to locate the general structure of the tumor tissue. IHC with Histone H3 is used as control. ( B ) qPCR analysis was used to observe p53 and HIF-1α gene expression in the ten selected regions, as shown in figure A. These 10 hypoxic regions were laser-captured, and gene expression was analyzed in triplicate. Normoxic regions were defined by negative HIF-1α staining; 5 such sites were selected, and gene expression was analyzed in triplicate. Blue and red arrows point towards easily observable trends for a simultaneous increase and decrease in HIF-1α and p53 expression, respectively. ( C ) Box plots present collective data from 10 different areas and analyzed in triplicates in figure B and show increased expression of both p53 (red) and HIF-1α (blue) in ten heterogeneous areas from single pancreatic and kidney cancer. Correlation coefficient analysis reveals R 2 = 0.6247 and R 2 = 0.0882 for pancreatic and kidney cancer, respectively ( n = 10 for hypoxic regions, n = 5 for normoxic regions; all samples were analyzed in triplicates). Two-sided Student's t -test was used for analysis; all P values are labeled on the figure.

Article Snippet: p53 WT cancer cells including A375, IM-9, HepG2, SKCO-1, HeLa, MCF-7, U-87-MG, LNCaP-FGC, NCI-H711, HCT116, U2OS and p53 MT cancer cells including MDA-MB-468, MOLT4, SW837, NCI-H23, P3HR1, PSN1, SKLMS1, SKLU1, SK-UT-1 and SNU-16 lines were procured from ATCC (VA, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Control, Gene Expression, Labeling

Journal: Nucleic Acids Research

Article Title: HIF-transcribed p53 chaperones HIF-1α

doi: 10.1093/nar/gkz766

Figure Lengend Snippet: HIF-1α correlates with both WT and MT p53 in hypoxic cells and hypoxic cancer tissues. ( A ) Box plots represent qRT-PCR-based quantification of p53 and HIF-1α gene expression in laser-captured hypoxic and normoxic regions of 6 different cancer types. For each cancer type, three different patient samples were collected, and from each sample, three different normoxic or hypoxic region were selected, thus for each cancer, three collected samples were analyzed in triplicates. The correlation coefficient between HIF-1α and p53 mRNA expression was calculated for each cancer, and R 2 values are presented in the plots. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure. ( B ) p53 and HIF-1α gene expressions were quantified by qRT-PCR analysis from a panel of 10 WT p53 and 10 MT p53 cell lines (listed in 2C) cultured under normoxia or exposed to hypoxia (1.8% O 2 ) for 24 h. For each cell line, gene expression was analyzed on three independent replicates per p53 WT or p53 MT cell lines. Correlation coefficient analysis shows a positive correlation between HIF-1α and p53 expression in MT p53 and WT p53 cell lines. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure. ( C ) Heat map depicts p53 and HIF-1α gene expression under normoxia and hypoxia for each WT p53 and MT p53 cancer cell line (quantified in 2C). HIF-1α and p53 expression increased in all cell lines under hypoxia, irrespective of p53 status. In this figure, each block represents the values from a gradient scale of low (red), medium (yellow) and high (blue) level of expression.

Article Snippet: p53 WT cancer cells including A375, IM-9, HepG2, SKCO-1, HeLa, MCF-7, U-87-MG, LNCaP-FGC, NCI-H711, HCT116, U2OS and p53 MT cancer cells including MDA-MB-468, MOLT4, SW837, NCI-H23, P3HR1, PSN1, SKLMS1, SKLU1, SK-UT-1 and SNU-16 lines were procured from ATCC (VA, USA).

Techniques: Quantitative RT-PCR, Gene Expression, Expressing, Labeling, Cell Culture, Blocking Assay

Journal: Nucleic Acids Research

Article Title: HIF-transcribed p53 chaperones HIF-1α

doi: 10.1093/nar/gkz766

Figure Lengend Snippet: Genetic manipulation of HIF-1α affects p53 expression. ( A ) Effect of HIF-1α on p53 expression in knockdown and overexpression experiments was observed using qRT-PCR in five p53 WT and five p53 MT cell lines (listed in 3B). Empty vector, HIF-1α -shRNA, non-specific Scr-shRNA, HIF-1α , or p53 -shRNA were transfected in normoxic or hypoxic (1.8% O 2 ) cultured cells for 24 hrs. Hypoxia-induced expression of p53 was abolished by HIF-1α -shRNA and increased upon exogenous addition of HIF-1α . For each cell line, gene expression was analyzed on three independent replicates ( n = 5, per p53 WT or MT p53 cell lines). Inset: Western blot analysis confirms the effective knockdown of p53 and HIF-1α in PSN1 cells. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure. ( B ) Heat map depicts p53 and HIF-1α gene expression under normoxia and hypoxia for each WT p53 and MT p53 cancer cell line (quantified in 3A). Results confirm effective knockdown of p53 in by p53 -shRNA, knockdown of HIF-1α by HIF-1α -shRNA, upregulation of HIF-1α by exogenous addition. Hypoxia-induced expression of WT or MT p53 was dependent on HIF-1α levels. In this figure, each block represents the values from a gradient scale of low (red), medium (yellow) and high (blue) level of expression ( n = 3). ( C ) Effect of HIF-1α knockdown and overexpression on p53 expression was observed by qRT-PCR in WT and HIF-1α KO [MCF-7 (WT p53 ) and PSN-1 (MT p53 )] hypoxic and normoxic cells. Crispr-generated HIF-1α null MCF-7 and PSN1 cells show no HIF-1α expression in normoxic and hypoxic cells (2 and 7). In hypoxic WT HIF-1α MCF-7 and PSN1 cells (panel 3–6), a consistent increase in p53 expression is observed with a dose-dependent increase in HIF-1α expression. All hypoxic cells were cultured for 24 h in 1.8% O 2 , n = 3, error bars represent SD. ( D ) Western blot analysis of whole-cell extracts from HIF-1α -WT MCF-7 or PSN1 and Crispr-generated HIF-1α -/− MCF-7 and PSN1 cells cultured under normoxia or hypoxia (at 1.8%O 2 ) for 24 h ( n = 3 independent replicates). HIF-1α protein was absent in all cell lines under normoxia and increased in hypoxic HIF-1α -WT cells, but not HIF-1α −/− cells (top panel). Hypoxia resulted in increased p53 expression in HIF-1α -WT cells, however, in hypoxic HIF-1α −/− cells, depleted of HIF-1 α protein, p53 protein does not increase compared to normoxic HIF-1α −/− cells (middle panel). β-actin was used as a loading control. ( E ) Western blot analysis of HIF-1α and p53 was performed using whole-cell extracts from HIF-1α WT and HIF-1α −/− MCF-7 cells treated with or without increasing amounts of HIF-1α and cultured under normoxia or hypoxia (at 1.8%O 2 ) for 24 h ( n = 3 independent replicates). HIF-1α and p53 protein increase in hypoxia-treated WT MCF-7 cells compared to normoxia. In HIF-1α −/− cells, hypoxia does not induce HIF1-α, and no significant increase in p53 is observed compared to normoxia (lane 7 versus lane 2). Exogenous addition of HIF-1α to hypoxic HIF-1α −/− cells restores HIF-1α expression and results in robust p53 induction in HIF-1 α dose-dependent manner (lanes 8–10). β-actin was used as the loading control.

Article Snippet: p53 WT cancer cells including A375, IM-9, HepG2, SKCO-1, HeLa, MCF-7, U-87-MG, LNCaP-FGC, NCI-H711, HCT116, U2OS and p53 MT cancer cells including MDA-MB-468, MOLT4, SW837, NCI-H23, P3HR1, PSN1, SKLMS1, SKLU1, SK-UT-1 and SNU-16 lines were procured from ATCC (VA, USA).

Techniques: Expressing, Knockdown, Over Expression, Quantitative RT-PCR, Plasmid Preparation, shRNA, Transfection, Cell Culture, Gene Expression, Western Blot, Labeling, Blocking Assay, CRISPR, Generated, Control

Journal: Nucleic Acids Research

Article Title: HIF-transcribed p53 chaperones HIF-1α

doi: 10.1093/nar/gkz766

Figure Lengend Snippet: HIF-1α transcriptionally regulates p53. ( A ) Luciferase assay was used to determine hypoxia-induced and HIF-1α-dependent activation of full-length p53 (10kb) promoter, a known HRE in VEGF promoter was cloned in PGL4 promoter and used as a positive control. Normoxia and hypoxia p53 WT MCF-7 cells were transfected with VEGF -PGL4-HRE or p53 10kb-PGL4 alone or co-transfected with HIF-1α -shRNA under and harvested for luciferase assay. A known p53 DBS in p21 promoter, p53 p21 –5′-DBS-pGL4 was used as a positive control in doxorubicin-treated normoxic MCF-7 cells. Empty vector or Beta-galactosidase (β-Gal) served as normalization controls; error bars represent S.D., n = 3. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure. ( B ) Luciferase assay was used to assess the activity of 5 putative HREs in the p53 promoter, as depicted in the model. p53 WT MCF-7 normoxic and hypoxic cells were transfected with VEGF -PGL4 vector, as a positive control for HIF-1α activity, or each p53 -HRE-PGL4 vectors with or without HIF-1α -shRNA. Doxo-treated MCF-7 positive cells transfected with p53 p21 -5′-DBS-pGL4 vector serves as positive control under normoxic conditions. The five p53 -HREs sites display hypoxia-inducible luciferase activity that is blocked by shRNA-mediated HIF-1α knockdown. Empty vector or Beta-galactosidase (β-Gal) served as normalization controls; error bars represent S.D., n = 3. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure. ( C ) Luciferase assay was used to assess the 5 HREs in p53 promoter under normoxia and hypoxia in HIF-1α -WT/KO MCF-7 and PSN1 cells. All cells were treated as described in (B) and harvested for luciferase assay. In both HIF1-α -WT MCF-7 (WT p53 ) and PSN1 (MT p53 ) cell lines, hypoxia leads to robust p53 promoter activity at each HRE (lanes 22–26, left and right panel) compared to normoxia. Hypoxic induction of HRE promoter activity is lost in HIF-1α −/− MCF-7 and PSN1 cells (lanes 31–35, left and right panel), indicating hypoxic induction of p53 promoter activity is HIF-1α-dependent. Empty vector or Beta-galactosidase (β-Gal) served as normalization controls; error bars represent S.D., n = 3. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure. ( D ) The ChIP-PCR assay was used to determine if HIF-1α binds to the five predicted HREs in p53 promoter in hypoxic WT PSN1 and HIF-1α −/− PSN1 cells. Lane 1: chromatin input. Lane 2: No Antibody negative control. Lane 3: a scrambled primer (Scr Pri) as a control for non-specific DNA PCR amplification. Lane 4: HIF-1α −/− cells as a negative control for HIF-1α protein binding. Lane 5: binding of HIF-1α protein to each of the 5 HREs is observed, n = 3. ( E ) Heat map depicts HIF-1α binding to the 5 HREs in p53 promoter measured by ChIP-qPCR in normoxic and hypoxic regions of kidney, pancreatic, colon, breast, lung and liver patient tumors. ChIP for HIF-1α binding to the VEGF -HRE was included as a positive control to confirm HIF-1α activation and binding activity. Variable HIF-1α binding to p53 -HREs was detected in hypoxic regions, but not normoxic regions, from all cancer types. In this figure, each block represents the values from a gradient scale of low (red), medium (yellow), and high (blue) level of expression, n = 3 (biological replicates). ( F ) Box plot summary of ChIP-qPCR enrichment for each HRE in cancer hypoxic regions as described in (E). Fold enrichment depicts HIF-1α binding to VEGF -HRE or HRE 1–5 in p53 promoter, in the hypoxic regions relative to HIF-1α binding in the normoxic regions, error bars represent S.D., n = 3.

Article Snippet: p53 WT cancer cells including A375, IM-9, HepG2, SKCO-1, HeLa, MCF-7, U-87-MG, LNCaP-FGC, NCI-H711, HCT116, U2OS and p53 MT cancer cells including MDA-MB-468, MOLT4, SW837, NCI-H23, P3HR1, PSN1, SKLMS1, SKLU1, SK-UT-1 and SNU-16 lines were procured from ATCC (VA, USA).

Techniques: Luciferase, Activation Assay, Clone Assay, Positive Control, Transfection, shRNA, Plasmid Preparation, Labeling, Activity Assay, Knockdown, Negative Control, Control, Amplification, Protein Binding, Binding Assay, ChIP-qPCR, Blocking Assay, Expressing

Journal: Nucleic Acids Research

Article Title: HIF-transcribed p53 chaperones HIF-1α

doi: 10.1093/nar/gkz766

Figure Lengend Snippet: Both MT and WT p53 binds to HIF-1α. ( A ) Left panels: Immunoprecipitation (IPP) assay of endogenous HIF-1α from the nuclear fraction of hypoxia-treated HIF-1α and p53 WT MCF7, HIF-1α WT and p53 MT PSN1 cells, HIF-1α KO MCF7 and PSN1 cells (Crispr-assisted) and HIF-1α and p53 -double KO MCF7 and PSN1 cells (Crispr-assisted). The top two panels are developed with Anti-HIF-1α Ab. The bottom two panels are developed with Anti-p53 Ab. Both HIF-1α and p53 bands are observed in WT cells of MCF-7 and PSN1 origin (first lane). No bands are observed in HIF-1α KO cells as IPP was performed using anti-HIF-1α Ab (lane 2). In p53 KO cells bands were observed IPP and development were performed using anti-HIF-1α Ab, and no bands were detected when IPP was performed using anti-HIF-1α Ab and development was performed using anti-p53 Ab (lane 3). In lane 4, no bands were observed as HIF-1α , and p53 double KO cells are used, and IPP is performed using anti-HIF-1α Ab. In the right panel: identical cell lines are used, but here the IPP is performed using anti-p53 Ab. In lane 1 bands are observed with development with both anti-p53 and anti-HIF-1α Abs. In lane 2 bands are observed in top 2 blots where p53 is IPPed and developed within HIF-1α KO cells, but when blots are developed with anti-HIF-1α Ab, these bands disappear. In lane 3 and 4, no bands are observed in p53 KO, and HIF-1α and p53 double KO cells as IPP is performed using anti-p53 Ab, n = 3. ( B ) Immunoprecipitation (IPP) assay of endogenous p53 and HIF-1α using the nuclear fraction (NF) from hypoxic tissue regions of 8 human cancers. In the first blot, IPP was performed against anti-p53 Ab and developed for HIF-1α protein; in second blot IPP was performed against anti-HIF-1α Ab and developed for p53 protein. HIF-1α and p53 were found to co-precipitate with each other in all hypoxic human cancer samples. In the third blot, all IPPs against HIF-1α show a positive signal for HIF-1 α protein, confirming IPP efficiency and antibody accuracy. In the fourth blot, IPP against RNAseH-II, a protein with no known p53 interaction, and developed against p53 results in no positive p53 signal for all samples, indicating accurate pull-down and antibody specificity for p53 protein. Positive detection of RNA-Pol II and negative detection of Tubulin were used as a protein loading control and for confirmation for the purity of nuclear fraction, n = 3. ( C ) Western blot analysis of p53 expression was performed using normoxic and hypoxic (1.8% O 2 for 24 h) p53 WT MCF-7-, U2OS- and HepG-2 cells. Hypoxia induces p53 expression in each of the cell lines compared to normoxia. β-actin was used as a loading control, n = 3. ( D ) In vivo , ELISA was conducted to observe conformation shift recognized by p53 Ab-1620 (p53 WT conformation) and p53 Ab-240 (p53 MT conformation). WT p53 MCF-7, UOS2, and HepG2 cells were cultured under normoxia and hypoxia (1.8% O 2 for 24 h). Hypoxia resulted in the loss of p53 Ab-1620 ELISA signal and increased p53 Ab-240 signal, indicating that hypoxia converts p53 to attain a mutant-like conformation, error bars represent S.D., n = 3. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure. ( E ) Luciferase assay was used to observed p53-mediated activation of its downstream p21 and Bax -minimal promoters. The p53-DBS within the promoter of the p21 and Bax genes, two known p53 gene targets, were cloned into the pGL4 vector. Normoxic and hypoxic doxorubicin-treated MCF-7 cells were transfected with p53 p21 -5′-DBS-pGL4 vector and with p53 Bax -5′-DBS-pGL4 and then harvested for luciferase assay. p53-driven transcriptional activity was lost under hypoxic conditions. Empty vector or Beta-galactosidase (β-Gal) served as normalization controls; error bars represent S.D., n = 3. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure.

Article Snippet: p53 WT cancer cells including A375, IM-9, HepG2, SKCO-1, HeLa, MCF-7, U-87-MG, LNCaP-FGC, NCI-H711, HCT116, U2OS and p53 MT cancer cells including MDA-MB-468, MOLT4, SW837, NCI-H23, P3HR1, PSN1, SKLMS1, SKLU1, SK-UT-1 and SNU-16 lines were procured from ATCC (VA, USA).

Techniques: Immunoprecipitation, CRISPR, Control, Western Blot, Expressing, In Vivo, Enzyme-linked Immunosorbent Assay, Cell Culture, Mutagenesis, Labeling, Luciferase, Activation Assay, Clone Assay, Plasmid Preparation, Transfection, Activity Assay

Journal: Nucleic Acids Research

Article Title: HIF-transcribed p53 chaperones HIF-1α

doi: 10.1093/nar/gkz766

Figure Lengend Snippet: WT and MT p53 enhance HIF-1α-dependent transcription and binding at HREs of downstream genes. ( A ) Luciferase activity was observed to study the impact of both WT and MT p53 on HIF-1α-dependent transcription at the minimal promoters of its downstream genes VEGF and EPO . Hypoxic (1.8% O 2 , 24 h) p53 WT MCF7 and p53 KO MCF7 cells were co-transfected with VEGF -PGL4 HRE or EPO -PGL4 HRE in the presence of p53 shRNA, WT p53 or MT p53 and then harvested for luciferase assay. Loss of p53 reduces HIF-1α-driven transcription at VEGF and EPO minimal promoters with HREs. Empty vector or Beta-galactosidase (β-Gal) served as normalization controls; error bars represent S.D., n = 3, Two-sided Student's t -test was used for analysis; all P values are labeled on the figure. ( B ) ChIP-PCR assay in hypoxia-treated p53 WT PSN1 and p53 KO PSN1 cells was performed to observe binding affinity of HIF-1α to the HREs in the VEGF and EPO promoters in presence and absence of WT and MT p53 . Lane 1; 2% input was used, lane 2; no Antibody (Ab) was used as IP control, lane 7; Scrambled primers (scr pri) included as PCR amplification control, lane 8; actin Ab is used as a negative control. Data shows reduced enrichment of both VEGF and EPO promoters in p53 KO PSN1 cells (lane 4) when compared to WT PSN1 cells (lane 3). Exogenous addition of WT p53 (lane 5) and MT p53 (lane 6) restores enrichment of HIF-1α at its HREs. ( C ) Heat map depicts qChIP assay demonstrating HIF-1α enrichment at the minimal promoters of 15 downstream genes, which are HIF targets with well-defined HREs. DNA was harvested from p53 WT MCF7, p53 WT HCT-116, p53 KO MCF7, MT p53 MC7 and MT p53 HCT116 cell line-derived xenografts grown in the flank region of the hind leg of nude mice. In addition, patient cancer samples from Kidney ( n = 4), pancreatic ( n = 3), and colon ( n = 3) tumors characterized by high, or low p53 expression were used. And finally, to observe if WT or MT p53 impacts HIF-1α enrichment at HREs WT p53 and MT p53 breast, colon and pancreatic patient cancer samples were analyzed. Row 1; 2% input was used in all samples, row 17; no Antibody (Ab) included as IP control, row 18; Scrambled primers (Scr Pri) included as PCR amplification control and row 19; as non-specific anti-actin Ab as, negative control. In this figure, each block represents the values from a gradient scale of low (red), medium (yellow) and high (blue) level of expression. HIF-1α-enrichment is significantly lower in hypoxic p53 null tumor xenografts (lane 3, 4), when compared with WT p53 (lanes 1, 2) and MT p53 (lanes 5, 6) tumor xenografts. In the patient samples hypoxic human tumors regions with high p53 expression, show increased enrichment of HIF-1α on its downstream HREs (lane 7–16), when compared to regions with low p53 expression (lanes 17–26). Finally, no significant difference in HIF-1α enrichment was observed between WT p53 (lane 27–29) and MT p53 (lane 30–32) tumor, n = 3. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure. ( D ) Top panel: Expression of 15 HIF-regulated genes described in A, were analyzed by qRT-PCR in hypoxic regions p53 WT MCF-7 and HCT-116, p53 −/− MCF-7 and HCT-116, and MT p53 expressing p53 −/− MCF-7 and HCT-116 tumor xenografts. Loss of p53 significantly reduced expression of all 15 genes. Data from MCF-7 and HCT tumors are pooled for analysis, n = 3, error bars represent S.D., n = 3. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure. Bottom panel: Expression of these genes is observed in pooled samples from hypoxic regions of four kidney, three pancreatic and four colon tumors characterized by high and low p53 expression (Inset below the plots). Reduced target gene expression was observed in hypoxic zones of cancers with low p53 expression compared to hypoxic zones with high p53 expression. Data from tumors with high versus low p53 expression is pooled for analysis, n = 3, error bars represent S.D., n = 3. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure.

Article Snippet: p53 WT cancer cells including A375, IM-9, HepG2, SKCO-1, HeLa, MCF-7, U-87-MG, LNCaP-FGC, NCI-H711, HCT116, U2OS and p53 MT cancer cells including MDA-MB-468, MOLT4, SW837, NCI-H23, P3HR1, PSN1, SKLMS1, SKLU1, SK-UT-1 and SNU-16 lines were procured from ATCC (VA, USA).

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, shRNA, Plasmid Preparation, Labeling, Control, Amplification, Negative Control, Derivative Assay, Expressing, Blocking Assay, Quantitative RT-PCR, Targeted Gene Expression

Journal: Nucleic Acids Research

Article Title: HIF-transcribed p53 chaperones HIF-1α

doi: 10.1093/nar/gkz766

Figure Lengend Snippet: Both WT and MT p53 form a transcriptional complex with HIF-1α at HREs. ( A ) Left panel: ChIP-PCR assay to observe if p53-HIF-1α complex binds to HREs of HIF downstream genes such as VEGF . In a genetically controlled experiment, WT p53 (HCT-116 and MCF-7), p53 KO MCF-7 and MT p53 A-431 cells were used. Lane 1; 2% input was used, lane 2; no Antibody (Ab) was used as IP control, lane 3; actin Ab is used as a negative control, lane 4; Scrambled primers (scr pri) included as PCR amplification control. ChIP with anti-p53 ab for HRE at VEGF minimal promoter gives a band in WT and MT p53 cells, suggesting that p53 binds to hypoxia response element, lane 5. ChIP with anti-HIF-1α Ab shows bands in all cell types, lane 6. In lanes 7 and 8, HIF-1α KD and use of HIF-1α KO cells (MCF-7 origin), results in disappearance for bands for both HIF-1α and p53 Abs, suggesting that p53 does not bind directly to HREs but is present there because of its binding to HIF-1α. In lanes 9 and 10, use of p53 shRNA or p53 KO MCF-7 cells abolishes all bands for ChIP with anti-p53 Ab, but the bands for ChIP with anti-HIF-1α Ab are present, suggesting that p53 is not required for HIF-1α to bind to its HREs, n = 3. In the second panel: this binding of p53 to HIF-1α response elements is observed in WT and MT hypoxic breast tumors, bands are observed for both anti-p53 and anti-HIF-1α ChIP, lanes 5 and 6 respectively, suggesting that HIF-1α-p53 complex is functional during HIF-driven transcription in hypoxic human cancers, n = 3. ( B ) ChIP-PCR assay using Anti-p53 Ab to measure p53 binding to the p53 DBS in the Bax promoter from normoxic and hypoxic zones of p53 WT and p53 MT breast cancer patient samples. Lane 1; 2% input was used, lane 2; no Antibody (Ab) was used as IP control, lane 3; anti-actin Ab was used as a negative control for ChIP, and lane 4; scrambled primers (Scr Pri) included as PCR amplification control. In lane 5; the data show that p53 binds to Bax p53-DBS only in the presence of WT p53 in normoxic tumors. In hypoxic tumors of any p53 origin or normoxic MT p53 tumors, this association is not observed, n = 3. ( C ) A model shows the mechanism where WT/MT p53 binds to HIF-1α and not to HREs directly.

Article Snippet: p53 WT cancer cells including A375, IM-9, HepG2, SKCO-1, HeLa, MCF-7, U-87-MG, LNCaP-FGC, NCI-H711, HCT116, U2OS and p53 MT cancer cells including MDA-MB-468, MOLT4, SW837, NCI-H23, P3HR1, PSN1, SKLMS1, SKLU1, SK-UT-1 and SNU-16 lines were procured from ATCC (VA, USA).

Techniques: Control, Negative Control, Amplification, Binding Assay, shRNA, Functional Assay

Journal: Nucleic Acids Research

Article Title: HIF-transcribed p53 chaperones HIF-1α

doi: 10.1093/nar/gkz766

Figure Lengend Snippet: Hypoxic WT & MT p53 chaperones HIF-1α at chromatin. ( A ) In vivo Chaperone Assay to measure the ability of WT or MT p53 to chaperone HIF-1α. The output read of this chaperone assay is the increase HIF-1α-driven transcription at its downstream VEGF HRE. p53 −/− MCF7 or p53 and HIF-1α double KO MCF-7 cells were co-transfected with [GAL4-BD] 5 - VEGF -HRE luciferase construct together with GAL4, WT p53 -GAL4, MT p53 -GAL4 alone or in combination with HIF-1α shRNA. In the left panel, this HIF-driven construct shows no activity in normoxia-cultured cells (lane-3–9), p53-driven p21 luciferase vector in doxorubicin-treated cells, is used as a positive control in normoxia (lane 10). Under hypoxic conditions, a baseline activity of the HIF-1α-driven VEGF promoter is observed (lane 13). Interestingly presence of WT p53 (lane 14) or MT p53 (lane 15) as chaperone increases HIF-1α-driven transcription; this effect is lost upon addition of HIF-1α shRNA or in HIF-1α KO cells (lane16–19). Empty vector or Beta-galactosidase (β-Gal) served as normalization controls; error bars represent S.D., n = 3. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure. ( B ) Western blot analysis of cycloheximide chase to study the effects of p53 expression on HIF-1α protein stability. p53 KO MCF-7 cells and p53 KO MCF-7 cells transfected with WT p53 or MT p53 were cultured under hypoxia for 24 h and treated with cycloheximide (100 μg/ml) for indicated times. HIF-1α protein is degraded within 6 h in p53 KO cells (lane 3), but both WT and MT p53 protect HIF-1 α against protein degradation (lanes 6 and 9). ( C ) Statistical analysis and quantification of the HIF-1α protein expression during the cycloheximide chase experiment shown in Figure is presented. Error bars represent S.D., n = 3. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure.

Article Snippet: p53 WT cancer cells including A375, IM-9, HepG2, SKCO-1, HeLa, MCF-7, U-87-MG, LNCaP-FGC, NCI-H711, HCT116, U2OS and p53 MT cancer cells including MDA-MB-468, MOLT4, SW837, NCI-H23, P3HR1, PSN1, SKLMS1, SKLU1, SK-UT-1 and SNU-16 lines were procured from ATCC (VA, USA).

Techniques: In Vivo, Transfection, Luciferase, Construct, shRNA, Activity Assay, Cell Culture, Plasmid Preparation, Positive Control, Labeling, Western Blot, Expressing

Journal: Nucleic Acids Research

Article Title: HIF-transcribed p53 chaperones HIF-1α

doi: 10.1093/nar/gkz766

Figure Lengend Snippet: Chaperoning of HIF-1α by WT & MT p53 promotes HIF-1 transcriptional activity. ( A ) In vitro transcription assay was performed using a cassette designed with six VEGF HRE repeats, followed by AdML core promoter, start codon of a G-less cassette and Poly-A tail. The RNA-Poll machinery and required transcription factor HIF-1α and molecular chaperone p53 were obtained from nuclear extracts of MCF-7 HIF-1α KO, p53 KO, and double HIF-1α p53 KO cells, and the readout of the assay was obtained by qPCR and by QIAXEL. In lane 1 no DNA template is added (negative control). No signal is observed where HIF-1α is lacking, lane 2–5. A signal is observed in from nuclear extract of MCF-7 HIF-1α and p53 double KO exogenously transfected with HIF-1α (lane 6), which is further amplified by rescuing WT or MT p53 expression (lanes 7–9). Exogenous addition of p53 N-terminus (A.A. 1–125) also rescues HIF-1α-driven transcription (lane 10). No signal is observed from normoxic cells because they lack HIF-1α expression (lane 11–12). Error bars represent S.D., n = 3. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure. ( B ) Effects of chaperone assay and in vitro transcription are observed in cellular conditions. Luciferase assay was performed on normoxic and hypoxic MCF-7 cells of different genetic backgrounds transfected with PGL4 luciferase vector with a VEGF HRE. Under hypoxia, cells carrying WT HIF-1α and WT p53 show a significant increase in VEGF HRE activity that is inhibited by loss of HIF-1α or p53 and augmented by overexpression of WT or MT p53 . Error bars represent S.D., n = 3. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure. ( C ) The effects of HIF-1α and p53 genetic manipulation were tested on a panel of 5 HIF-1α downstream genes, EPO, GPI, SERPINE1, VEGFA , and NDRG1 . Genetically manipulated normoxic or hypoxic MCF-7 cells were used for qRT-PCR analysis. Heat map depicts the gene expression of HIF-1α target genes. HIF-1α-regulated genes are poorly expressed under normoxia and highly induced by hypoxia plus overexpression of WT or MT p53 in HIF +/+ ; p53 −/− cells. p53-induced upregulation of HIF-1α target genes is diminished by concurrent HIF-1α deficiency. In this figure, each block represents the values from a gradient scale of low (red), medium (yellow) and high (blue) level of expression, n = 3. Two-sided Student's t -test was used for analysis; all P values are labeled on the figure.

Article Snippet: p53 WT cancer cells including A375, IM-9, HepG2, SKCO-1, HeLa, MCF-7, U-87-MG, LNCaP-FGC, NCI-H711, HCT116, U2OS and p53 MT cancer cells including MDA-MB-468, MOLT4, SW837, NCI-H23, P3HR1, PSN1, SKLMS1, SKLU1, SK-UT-1 and SNU-16 lines were procured from ATCC (VA, USA).

Techniques: Activity Assay, In Vitro, Transcription Assay, Negative Control, Transfection, Amplification, Expressing, Labeling, Luciferase, Plasmid Preparation, Over Expression, Quantitative RT-PCR, Gene Expression, Blocking Assay

Journal: Nucleic Acids Research

Article Title: HIF-transcribed p53 chaperones HIF-1α

doi: 10.1093/nar/gkz766

Figure Lengend Snippet: Model depiction: Positive feedback loop between HIF-1α and p53 drives HIF-1α downstream signaling. Hypoxia-stimulated HIF-1α binds to HREs within p53 promoter and increases p53 transcription resulting in p53 protein accumulation. Under hypoxia, p53 may obtain a MT conformation. Both MT and WT p53 bind to HIF-1α protects HIF-1 α against protein degradation and chaperones HIF-1α toward the response elements of target genes, increasing transcriptional activity. Ultimately, p53-chaperoning of HIF-1 α under hypoxia exacerbates HIF-1 α signaling, fueling the pro-tumorigenic properties of HIF-1α.

Article Snippet: p53 WT cancer cells including A375, IM-9, HepG2, SKCO-1, HeLa, MCF-7, U-87-MG, LNCaP-FGC, NCI-H711, HCT116, U2OS and p53 MT cancer cells including MDA-MB-468, MOLT4, SW837, NCI-H23, P3HR1, PSN1, SKLMS1, SKLU1, SK-UT-1 and SNU-16 lines were procured from ATCC (VA, USA).

Techniques: Activity Assay

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: 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: Cell lines and culture conditions 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: Cell lines and culture conditions 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: Cell lines and culture conditions 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: Cell lines and culture conditions 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: Cell lines and culture conditions 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