Distinct mechanisms control genome recognition by p53 at its target genes linked to different cell fates.

The tumor suppressor p53 integrates stress response pathways by selectively engaging one of several potential transcriptomes, thereby triggering cell fate decisions (e.g., cell cycle arrest, apoptosis). Foundational to this process is the binding of tetrameric p53 to 20-bp response elements (REs) in the genome (RRRCWWGYYYN0-13RRRCWWGYYY). In general, REs at cell cycle arrest targets (e.g. p21) are of higher affinity than those at apoptosis targets (e.g., BAX). However, the RE sequence code underlying selectivity remains undeciphered. Here, we identify molecular mechanisms mediating p53 binding to high- and low-affinity REs by showing that key determinants of the code are embedded in the DNA shape. We further demonstrate that differences in minor/major groove widths, encoded by G/C or A/T bp content at positions 3, 8, 13, and 18 in the RE, determine distinct p53 DNA-binding modes by inducing different Arg248 and Lys120 conformations and interactions. The predictive capacity of this code was confirmed in vivo using genome editing at the BAX RE to interconvert the DNA-binding modes, transcription pattern, and cell fate outcome

Multiple Breast Cancer Cell-Lines Derived from a Single Tumor Differ in Their Molecular Characteristics and Tumorigenic Potential

Background Breast cancer cell lines are widely used tools to investigate breast cancer biology and to develop new therapies. Breast cancer tissue contains molecularly heterogeneous cell populations. Thus, it is important to understand which cell lines best represent the primary tumor and have similarly diverse phenotype. Here, we describe the development of five breast cancer cell lines from a single patient’s breast cancer tissue. We characterize the molecular profiles, tumorigenicity and metastatic ability in vivo of all five cell lines and compare their responsiveness to 4-hydroxytamoxifen (4-OHT) treatment. Methods Five breast cancer cell lines were derived from a single patient’s primary breast cancer tissue. Expression of different antigens including HER2, estrogen receptor (ER), CK8/18, CD44 and CD24 was determined by flow cytometry, western blotting and immunohistochemistry (IHC). In addition, a Fuorescent In Situ Hybridization (FISH) assay for HER2 gene amplification and p53 genotyping was performed on all cell lines. A xenograft model in nude mice was utilized to assess the tumorigenic and metastatic abilities of the breast cancer cells. Results We have isolated, cloned and established five new breast cancer cell lines with different tumorigenicity and metastatic abilities from a single primary breast cancer. Although all the cell lines expressed low levels of ER, their growth was estrogen-independent and all had high-levels of expression of mutated non-functional p53. The HER2 gene was rearranged in all cell lines. Low doses of 4-OHT induced proliferation of these breast cancer cell lines. Conclusions All five breast cancer cell lines have different antigenic expression profiles, tumorigenicity and organ specific metastatic abilities although they derive from a single tumor. None of the studied markers correlated with tumorigenic potential. These new cell lines could serve as a model for detailed genomic and proteomic analyses to identify mechanisms of organ-specific metastasis of breast cancer

Skp2B Overexpression Alters a Prohibitin-p53 Axis and the Transcription of PAPP-A, the Protease of Insulin-Like Growth Factor Binding Protein 4

We previously reported that the degradation of prohibitin by the SCF(Skp2B) ubiquitin ligase results in a defect in the activity of p53. We also reported that MMTV-Skp2B transgenic mice develop mammary gland tumors that are characterized by an increased proteolytic cleavage of the insulin-like growth factor binding protein 4 (IGFBP-4), an inhibitor of IGF signaling. However, whether a link exists between a defect in p53 activity and proteolysis of IGFBP-4 was not established.We analyzed the levels of pregnancy-associated plasma protein A (PAPP-A), the protease of IGFBP-4, in MMTV-Skp2B transgenic mice and found that PAPP-A levels are elevated. Further, we found a p53 binding site in intron 1 of the PAPP-A gene and that both wild type and mutant p53 bind to this site. However, binding of wild type p53 results in the transcriptional repression of PAPP-A, while binding of mutant p53 results in the transcriptional activation of PAPP-A. Since MMTV-Skp2B mice express wild type p53 and yet show elevated levels of PAPP-A, at first, these observations appeared contradictory. However, further analysis revealed that the defect in p53 activity in Skp2B overexpressing cells does not only abolish the activity of wild type of p53 but actually mimics that of mutant p53. Our results suggest that in absence of prohibitin, the half-life of p53 is increased and like mutant p53, the conformation of p53 is denatured.These observations revealed a novel function of prohibitin as a chaperone of p53. Further, they suggest that binding of denatured p53 in intron 1 causes an enhancer effect and increases the transcription of PAPP-A. Therefore, these findings indicate that the defect in p53 function and the increased proteolysis of IGFBP-4, we had observed, represent two components of the same pathway, which contributes to the oncogenic function of Skp2B

Mdm2 promotes Cdc25C protein degradation and delays cell cycle progression through the G2/M phase

Upon different types of stress, the gene encoding the mitosis-promoting phosphatase Cdc25C is transcriptionally repressed by p53, contributing to p53’s enforcement of a G2 cell cycle arrest. In addition, Cdc25C protein stability is also decreased following DNA damage. Mdm2, another p53 target gene, encodes a ubiquitin ligase that negatively regulates p53 levels by ubiquitination. Ablation of Mdm2 by siRNA led to an increase in p53 protein and repression of Cdc25C gene expression. However, Cdc25C protein levels were actually increased following Mdm2 depletion. Mdm2 is shown to negatively regulate Cdc25C protein levels by reducing its half-life independently of the presence of p53. Further, Mdm2 physically interacts with Cdc25C and promotes its degradation through the proteasome in a ubiquitin-independent manner. Either Mdm2 overexpression or Cdc25C downregulation delays cell cycle progression through the G2/M phase. Thus, the repression of the Cdc25C promoter by p53, together with p53-dependent induction of Mdm2 and subsequent degradation of Cdc25C, could provide a dual mechanism by which p53 can enforce and maintain a G2/M cell cycle arrest.Fil: Giono, Luciana Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Resnick Silverman, Lois. Icahn School Of Medicine At Mount Sinai; Estados UnidosFil: Carvajal, Luis A.. Icahn School Of Medicine At Mount Sinai; Estados UnidosFil: St. Clair, Selvon. Icahn School Of Medicine At Mount Sinai; Estados UnidosFil: Manfredi, James J.. Icahn School Of Medicine At Mount Sinai; Estados Unido

p53 regulates PAPP-A levels.

<p>A) HBL-100 cells express wild type p53, while HS578T cells express mutant p53. The levels of PAPP-A mRNA in these cell lines were determined by qRT-PCR. B) Western blot of the levels of p53 in HBL-100 and HS578T cells. C) the expression of p53 was inhibited in HBL-100 cells using siRNA and the PAPP-A mRNA levels were determined by qRT-PCR. D) the efficiency of siRNA against p53 was determined by western blot. E) the expression of p53 was inhibited in HS578T cells using siRNA and the PAPP-A mRNA levels were determined by qRT-PCR. F) the inhibition of p53 by siRNA in HS578T cells was monitored by western blot. G) Duplicate human breast carcinoma tissue microarray slides (Zymed) were stained by IHC using the DO1 antibody against p53 and the rabbit polyclonal antibody against PAPP-A (Dako). H) The percentage of tumors positive for p53, PAPP-A or both was determined.</p

Skp2B overexpression promotes the accumulation of denatured p53 species.

<p>A) Cycloheximide chase in MCF-7 and MCF-7Skp2B cells was performed for the indicated time and cells were harvested for immunoblotting using anti-p53 antibody. Tubulin was used as a loading control. B) Quantification of the fraction of p53 remaining in MCF-7 and MCF-7Skp2B cells at the indicated time of cycloheximide treatment. C) Proteins from MCF-7, MCF-7Skp2B and T47D cells were used for immunoprecipitation of endogenous p53 using the mouse monoclonal pAb240 anti-p53 antibody, which immunoprecipitates only denatured but not wild-type forms of p53. The immunoblot was then developed using an anti-rabbit p53 antibody. D) Extracts from MCF-7 and MCF-7Skp2B were digested with increasing concentrations of thermolysin and the resulting cleavage products detected by immunoblot using the DO1 antibody. The 27kDa product is indicated. E) MCF-7 and MCF-7-Skp2B cells were treated with or without camptothecin (10 µM) for 4 hours and the levels of GADD45 mRNA levels determined qRT-PCR. The difference between the levels of GADD45 mRNA following DNA damage between MCF-7 and MCF7-Skp2B cells was statistically significant. The three arrows indicate a p value of less than 0.0001. F) MCF-7 and MCF-7-Skp2B cells were treated with or without camptothecin (10 µM) for 4 hours and the levels of PUMA mRNA levels determined qRT-PCR. The difference between the levels of PUMA mRNA following DNA damage between MCF-7 and MCF7-Skp2B cells was statistically significant. The three arrows indicate a p value of less than 0.0001. G) MCF-7 treated with mock siRNA and MCF-7-Skp2B cells were treated with 1 or 2 uM camptothecin for 7 days and the percentage of cells that survived determined. The difference between the percentage of cell surviving following DNA damage between MCF-7 and MCF7-Skp2B cells was statistically significant. The three arrows indicate a p value of less than 0.0001.</p

p53 regulates PAPP-A levels by binding to the PAPP-A promoter.

<p>A) The mammary glands from wild type, pregnant (wild type) MMTV-Skp2B and tumors were analyzed for the mRNA levels of PAPP-A by quantitative RT-PCR. B) The level of IGFBP-4 in the mammary gland of wild type mice that are either virgin, pregnant, lactating or involuting was determined by western blot. C) The estrogen receptor null breast cancer cells MDA-MB-157 were transfected with 3 ug of estrogen receptor plasmid and D) after 24 hours cells were harvested for analyzing the expression of the estrogen receptor and mRNA levels of PAPP-A by quantitative RT-PCR. E) The p53 null MDA-MB157 cells were transfected with 2 µg of wild type p53 and PAPP-A mRNA levels determined. F) The level of expression of transfected wild type p53 was determined by western blot. G) MDA-MB 157 cells were transfected with 2 µg of different mutants of p53 (p53–143 and p53–248), 24 hours after, cells were harvested for analysis of PAPP-A mRNA. H) Western blot showing the expression of mutant p53. Tubulin was used as a loading control. I) p53 binding site on PAPP-A promoter. MDA-MB157 cells transfected with 2 µg of wild type and mutant p53 constructs. After 24 hours of transfection cells were fixed with formaldehyde and collected for chromatin immunoprecipitation (ChIP) using the p53 (DO-1) antibody. Normal mouse IgG served as a negative control.</p