Exploring structural determinants and the role of nucleolin in formation of the long-range interactions between untranslated regions of p53 mRNA.

p53 protein is a key regulator of cellular homeostasis by coordinating the framework of antiproliferative pathways as a response to various stress factors. Although the main mechanism of stress-dependent induction of p53 protein relies on post-translational modifications influencing its stability and activity, a growing amount of evidence suggests that complex regulation of p53 expression occurs also at the mRNA level. This study explores structural determinants of long-range RNA-RNA interactions in p53 mRNA, crucial for stress-dependent regulation of p53 protein translation. We demonstrate that the 8-nt bulge motif plays a key structural role in base-pairing of complementary sequences from the 5' and 3' untranslated regions of p53 mRNA. We also show that one of the p53 translation regulators, nucleolin, displays an RNA chaperone activity and facilitates the association of sequences involved in the formation of long-range interactions in p53 mRNA. Nucleolin promotes base-pairing of complementary sequences through the bulge motif, because mutations of this region reduce or inhibit pairing while compensatory mutations restore this interaction. Mutational analysis of nucleolin reveals that all four RNA recognition motifs are indispensable for optimal RNA chaperone activity of nucleolin. These observations help to decipher the unique mechanism of p53 protein translation regulation pointing to bulge motif and nucleolin as the critical factors during intramolecular RNA-RNA recognition in p53 mRNA.

A Sub-Group of Kidney-Transplant Recipients with Highly Aggressive Squamous Cell Carcinoma Expressing Phosphorylated Serine392p53.

Cutaneous squamous cell carcinomas in kidney-transplant recipients are frequent, with an increasing incidence linked to long immunosuppression durations and exposure to ultraviolet radiation. p53 is at the cornerstone of ultraviolet-induced DNA damage, but the role of p53 post-translational modifications in this context is not yet deciphered. Here, we investigated the phosphorylation status of p53 at Serine 392 in 25 cutaneous squamous cell carcinomas in kidney-transplant recipients, compared with 22 non-transplanted patients. Cutaneous squamous cell carcinomas in transplanted patients occurred after a median period of 19 years of immunosuppression, with a median number of 15 cutaneous squamous cell carcinomas and more aggressive histological and clinical characteristics. There was no significant difference between Ki67, p53, and pSer392p53 expression in the two groups. Using principal component analysis, we identified a cluster of exclusively transplanted patients with a median of 23 years of immunosuppression duration, significantly more aggressive biological characteristics, and higher pSer392p53 expression. pSer392p53 was expressed in the whole tumor, suggesting an early carcinogenic event in the course of prolonged immunosuppression. This high, diffuse pSer392p53 expression, corresponding to a high level of DNA damage, might be useful to identify aggressive cutaneous squamous cell carcinomas in kidney-transplant recipients to treat them more aggressively.

Polypeptide Bilayer Assembly-Mediated Gene Delivery Enhances Chemotherapy in Cancer Cells.

Developing gene vectors with high transfection efficiency and low cytotoxicity to humans is crucial to improve gene therapy outcomes. This study set out to investigate the use of cationic polypeptide bilayer assemblies formed by coil-sheet poly(l-lysine)-block-poly(l-benzyl-cysteine) (PLL-b-PBLC) as gene vectors that present improved transfection efficiency, endosomal escape, and biocompatibility compared to PLL. The formation of the polyplexes was triggered by hydrogen bonding, hydrophobic interactions, and electrostatic association between the cationic PLL segments and the negatively charged plasmid encoding p53, resulting in self-assembled polypeptide chains. Transfection efficiency of these polyplexes increased with increments of PLL-to-PBLC block ratios, with PLL15-b-PBLC5 bilayers exhibiting the best in vitro transfection efficiency among all, suggesting that PLL-b-PBLC bilayer assemblies are efficient in the protection and stabilization of genes. The polypeptide bilayer gene vector reversed the cisplatin sensitivity of p53-null cancer cells by increasing apoptotic signaling. Consistent with in vitro results, mouse xenograft studies revealed that PLL15-b-PBLC5/plasmid encoding p53 therapy significantly suppressed tumor growth and enhanced low-dose cisplatin treatment, while extending survival of tumor-bearing mice and avoiding significant body weight loss. This study presents a feasible gene therapy that, combined with low-dose chemotherapeutic drugs, may treat genetically resistant cancers while reducing side effects in clinical patients.

Nanostructured poly(thiophene acetic acid)/Au/poly(methylene blue) interface for electrochemical immunosensing of p53 protein.

A poly(thiophene acetic acid)/Au/poly(methylene blue) nanostructured interface was electrochemically assembled step-by-step on screen-printed carbon electrodes (SPCE) for label-free detection of p53 protein. The initial electrical conductive properties of the polymeric interface were increased with an additional layer of poly(methylene blue) electropolymerized in the presence of gold nanoparticles. The nano-immunosensing architecture was prepared by covalent immobilization of anti-p53 antibodies as bioreceptors through the poly(thiophene acetic acid) moieties. The nano-immunosensor assembly was extensively characterized by ultraviolet-visible spectrophotometry, dynamic and electrophoretic light scattering, scanning electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, atomic force microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. Under optimal conditions, p53 was specifically and selectively detected by square wave voltammetry in a linear range between 1 and 100 ng mL-1 with a limit of detection of 0.65 ng mL-1. In addition, the electrochemical nano-immunosensor detected p53 in spiked human serum samples and colorectal cancer cell lysates, and the results were validated with a standard spectrophotometric method using a paired samples t test, which did not exhibit significant differences between both methods. The resultant p53 nano-immunosensor is simple to assemble, robust, and has the potential for point-of-care biomarker detection applications.

Low-frequency collective motion of DNA-binding domain defines p53 function.

Most mutations in the DNA-binding domain (DBD) of p53 inactivate or rescue the protein function interacting with the minor groove of DNA. However, how the conformation changes propagating from the mutation sites result in distinct molecular recognition is still not well understood. As the protein mobility is an intrinsic property encrypted in its primary structure, we examined if different structures of wild-type and mutant p53 core domains display any unique patterns of intrinsic mobility. Normal mode calculation was employed to characterize the collective dynamics of DBD in p53 monomer and tetramer as well as their mutants. Intriguingly, the low-frequency collective motions of DBD show similar patterns between the wild-type protein and the rescued mutants. The analysis on atomic backbone fluctuations and low-frequency vibration mode statistics does further support the correlation between the intrinsic collective motion of DBD and the p53 protein function. The mutations in the DBD influence the low-frequency vibration of the p53 tetramer via the change of the collective motions among its four monomers. These findings thus provide new insights for understanding the physical mechanism of p53 protein structure-function relationship and help find the small molecule drug to modulate protein dynamic for disease therapy.

A comprehensive study of p53 protein.

The protein p53 has been extensively investigated since it was found 43 years ago and has become a "guardian of the genome" that regulates the division of cells by preventing the growth of cells and dividing them, that is, inhibits the development of tumors. Initial proof of protein existence by researchers in the mid-1970s was found by altering and regulating the SV40 big T antigen termed the A protein. Researchers demonstrated how viruses play a role in cancer by employing viruses' ability to create T-antigens complex with viral tumors, which was discovered in 1979 following a viral analysis and cancer analog research. Researchers later in the year 1989 explained that in Murine Friend, a virus-caused erythroleukemia, commonly found that p53 was inactivated to suggest that p53 could be a "tumor suppressor gene." The TP53 gene, encoding p53, is one of human cancer's most frequently altered genes. The protein-regulated biological functions of all p53s include cell cycles, apoptosis, senescence, metabolism of the DNA, angiogenesis, cell differentiation, and immunological response. We tried to unfold the history of the p53 protein, which was discovered long back in 1979, that is, 43 years of research on p53, and how p53's function has been developed through time in this article.

A computer-aided drug design approach to discover tumour suppressor p53 protein activators for colorectal cancer therapy.

Colorectal cancer (CRC) is the third most detected cancer and the second foremost cause of cancer deaths in the world. Intervention targeting p53 provides potential therapeutic strategies, but thus far no p53-based therapy has been successfully translated into clinical cancer treatment. Here we developed a Quantitative Structure-Activity Relationships (QSAR) classification models using empirical molecular descriptors and fingerprints to predict the activity against the p53 protein, using the potency value with the active or inactive label, were developed. These models were built using in total 10,505 molecules that were extracted from the ChEMBL, ZINC and Reaxys® databases, and recent literature. Three machine learning (ML) techniques e.g., Random Forest, Support Vector Machine, Convolutional Neural Network were explored to build models for p53 inhibitor prediction. The performances of the models were successfully evaluated by internal and external validation. Moreover, based on the best in silico p53 model, a virtual screening campaign was carried out using 1443 FDA-approved drugs that were extracted from the ZINC database. A list of virtual screening hits was assented on base of some limits established in this approach, such as: (1) probability of being active against p53; (2) applicability domain; (3) prediction of the affinity between the p53, and ligands, through molecular docking. The most promising according to the limits established above was dihydroergocristine. This compound revealed cytotoxic activity against a p53-expressing CRC cell line with an IC50 of 56.8 µM. This study demonstrated that the computer-aided drug design approach can be used to identify previously unknown molecules for targeting p53 protein with anti-cancer activity and thus pave the way for the study of a therapeutic solution for CRC.

Inhibitor of apoptosis-stimulating p53 protein protects against inflammatory bowel disease in mice models by inhibiting the nuclear factor kappa B signaling.

Drugs and therapies available for the treatment of inflammatory bowel disease (IBD) are not satisfactory. Our previous study has established the inhibitor of apoptosis-stimulating p53 protein (iASPP) as an oncogenic regulator in colorectal cancer by forming a regulatory axis or feedback loop with miR-124, p53, or p63. As iASPP could target and inhibit nuclear factor kappa B (NF-κB) activation, in this study the role and mechanism of iASPP in IBD was investigated. The aberrant up-regulation of iASPP in IBD was subsequently confirmed, based on online data sets, clinical sample examinations and 2,4,6-trinitrobenzene sulfonic acid (TNBS)- and dextran sulfate sodium (DSS)-induced colitis mice models. TNBS or DSS stimulation successfully induced colon shortness, body weight loss, mice colon oxidative stress and inflammation. In both types of colitis mice models, iASPP over-expression improved, whereas iASPP knockdown aggravated TNBS or DSS stimulation-caused colon shortness, body weight loss and mice colon oxidative stress and inflammation. Meanwhile, in both types of colitis mice models, iASPP over-expression inhibited p65 phosphorylation and decreased the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, C-X-C motif chemokine ligand (CXCL)1 and CXCL2 in mice colons, whereas iASPP knockdown exerted opposite effects.

Characterizing TP53 mutations in ovarian carcinomas with and without concurrent BRCA1 or BRCA2 mutations.

OBJECTIVES: Mutations in the TP53 tumor suppressor gene are common in ovarian carcinoma (OC) but their impact on outcomes is controversial. We sought to define the relationship of TP53 mutations to cancer outcomes and their interactions with co-occurrent BRCA1 or BRCA2 (BRCA) mutations, comparing three different TP53 mutation classification schemes. METHODS: We performed next generation sequencing on 393 cases of OC prospectively followed for survival. TP53 mutations were classified according to three schemes termed Structural, Functional, and Hotspot. Mutation distribution was compared between cases with and without BRCA mutations. In a subset of 281 cases of high grade serous carcinoma (HGSC), overall survival was compared using Kaplan-Meier curves, logrank testing, and multivariate Cox regression analysis, both stratified and adjusted for BRCA mutation status. Multivariate logistic regression was used to analyze the effects of TP53 mutation type on platinum resistance. RESULTS: TP53 mutations were identified in 76.8% of the total cohort (n = 302/393) and 87.9% of HGSC (n = 247/281). Cases with BRCA mutations demonstrated significantly higher TP53 mutation frequency overall (n = 84/91, 92.3% vs. n = 218/302, 72.2%, p < 0.001). TP53 mutations were not associated with overall survival, even when stratified by BRCA mutation. TP53 mutations were associated with platinum sensitivity, even after adjusting for BRCA mutation status (OR 0.41, p = 0.048). The choice of TP53 mutation classification scheme was not found to alter any significant outcome. CONCLUSIONS: BRCA mutations significantly co-occur with TP53 mutations. After adjusting for BRCA mutations, TP53 mutations are associated with platinum sensitivity, and this effect is not dependent on TP53 mutation type.

Effect of pharmacodynamical interaction between nutlin-3a and aspirin in the activation of p53.

BACKGROUND AND OBJECTIVE: p53, an anti-tumour protein, is significantly inactivated in most tumours. A small molecule of nutlin-3a is used to activate its function by repressing (Mouse double minute 2 homolog) Mdm2 protein which inhibits its activity. In cancer patients, a high risk of drug-drug interactions (DDIs) is observed owing to their multi-dosing prescriptions, which may lead them to harmful effects. In the presented work, we have aimed to investigate the effect of pharmacodynamical interaction between two anti-cancer drugs, nutlin-3a and aspirin in the activation of p53 protein. METHODS: We have adapted control system techniques and designed a Proportional-Integral-Derivative (PID) controller. This controller is used to activate p53 protein. A drug interaction parameter is used to incorporate the effect of both drugs. Extensive simulation is performed using two different doses of aspirin, i.e. a low and a high dose of aspirin. RESULTS: The result shows no harmful effects of pharmacodynamical interaction when a low dose is administered along with nutlin-3a. When a high dose of aspirin is administered it acts as input disturbance and leads to undesirable over-expression of p53 protein. This can further harm other growth cells, thus inducing harmful effects. A comparative analysis is also tabulated with different dosing regimens which shows that a combination of nutlin-3a and a low dose of aspirin provides better results than a high dose of aspirin. CONCLUSION: Overall, the work provides an insight to the activation of p53 protein in cancer patients under the presence of pharmacodynamical interaction and might contribute to the effective management of cancer patients.

Solution structure of the anticancer p28 peptide in biomimetic medium.

The p28 peptide derived from Pseudomonas aeruginosa azurin shows an anticancer activity after binding to p53 protein and is currently in Phase I of clinical trials. We have studied its structure in water and in a biomimetic media and show that the peptide is unstructured in water but when studied in a biomimetic medium assumes a structure very similar to the one observed in azurin, suggesting a high propensity of this peptide to maintain this secondary structure. Analysis of p28 sequences from different bacterial species indicates conservation of the secondary structure despite amino acid replacement in different positions, suggesting that others, similar peptides could be tested for binding to p53.

Poly(C)-binding Protein 2 Regulates the p53 Expression via Interactions with the 5'-Terminal Region of p53 mRNA.

The p53 protein is one of the major transcriptional factors which guards cell homeostasis. Here, we showed that poly(C)-binding protein 2 (PCBP2) can bind directly to the 5' terminus of p53 mRNA by means of electrophoretic mobility shift assay. Binding sites of PCBP2 within this region of p53 mRNA were mapped using Pb2+-induced cleavage and SAXS methods. Strikingly, the downregulation of PCBP2 in HCT116 cells resulted in a lower level of p53 protein under normal and stress conditions. Quantitative analysis of p53 mRNA in PCBP2-downregulated cells revealed a lower level of p53 mRNA under normal conditions suggesting the involvement of PCBP2 in p53 mRNA stabilisation. However, no significant change in p53 mRNA level was observed upon PCBP2 depletion under genotoxic stress. Moreover, a higher level of p53 protein in the presence of rapamycin or doxorubicin and the combination of both antibiotics was noticed in PCBP2-overexpressed cells compared to control cells. These observations indicate the potential involvement of PCBP2 in cap-independent translation of p53 mRNA especially occurring under stress conditions. It has been postulated that the PCBP2 protein is engaged in the enhancement of p53 mRNA stability, probably via interacting with its 3' end. Our data show that under stress conditions PCBP2 also modulates p53 translation through binding to the 5' terminus of p53 mRNA. Thus PCBP2 emerges as a double-function factor in the p53 expression.

Photodynamic Therapy of Sarcoma M-1 in Rats with Photosensitizer Photoran E6.

We studied the effectiveness of photodynamic therapy with the photosensitizer Photoran E6 on the model of rat sarcoma M-1 positive for mutant p53 gene. Experiments showed that Photoran E6 exhibits high antitumor activity in photodynamic therapy of solid tumor of the connective tissue. Photodynamic therapy carried out during the optimal period after injections of Photoran E6 with the determined parameters of laser exposure allows achieving the maximum inhibitory effect on sarcoma M-1: 100% cured animals. Immunohistochemical study revealed no live tumor cells with expression of the mutant p53 protein in areas of photodynamic exposure.

Influence of Surgical Trauma in the Nasal Cavity on the Expression of p53 Protein in the Hippocampus of Rats.

The aim of the study was to determine the role of the stress effect of septoplasty modeling on p53 protein expression in the hippocampus of rats under conditions of sensory olfactory deprivation. Simulation of septoplasty was carried out on 30 sexually mature male rats. A quantitative assessment of the apoptosis of neurons in the pyramidal layer of the hippocampus in the subfields CA1, CA2, CA3, and dentate gyrus (DG) on days 2, 4, and 6 after surgery was carried out. Histological sections were stained by the immunohistochemical method with antibodies to the p53 protein. An increase in the number of p53-positive neurons was noted in all subfields; the maximum increase in the number of apoptotic neurons was noted on day 4 after surgery. The stress effect of modeling septoplasty in rats, accompanied by sensory deprivation of the peripheral part of the olfactory analyzer, provoked the expression of p53 and the initiation of apoptosis mechanisms in various subfields of the hippocampus.

Small-molecule modulation of p53 protein-protein interactions.

Small-molecule modulation of protein-protein interactions (PPIs) is a very promising but also challenging area in drug discovery. The tumor suppressor protein p53 is one of the most frequently altered proteins in human cancers, making it an attractive target in oncology. 14-3-3 proteins have been shown to bind to and positively regulate p53 activity by protecting it from MDM2-dependent degradation or activating its DNA binding affinity. PPIs can be modulated by inhibiting or stabilizing specific interactions by small molecules. Whereas inhibition has been widely explored by the pharmaceutical industry and academia, the opposite strategy of stabilizing PPIs still remains relatively underexploited. This is rather interesting considering the number of natural compounds like rapamycin, forskolin and fusicoccin that exert their activity by stabilizing specific PPIs. In this review, we give an overview of 14-3-3 interactions with p53, explain isoform specific stabilization of the tumor suppressor protein, explore the approach of stabilizing the 14-3-3σ-p53 complex and summarize some promising small molecules inhibiting the p53-MDM2 protein-protein interaction.

Detection of tumor suppressor protein p53 with special emphasis on biosensors: A review.

Cancer is a general word, which specifies a cluster of diseases affecting almost every-body part. Cancer is a second leading cause of death, globally. The tumor suppressor protein p53 is known to play a vital role in prevention of cancer. The enhanced and active form of p53 controls target gene expression through binding with DNA response elements (REs) and thus inhibits tumor cell growth. p53 is found mutated in more than 50% of the cancers. The wide mutation spectrum of p53 gene underlies the process of tumor development. Hence, the accurate quantification of p53 protein levels has great importance in early diagnosis of cancer. The biosensors are the tools, which convert biological interactions into readout signals. These are the most simple, sensitive, specific, rapid and precise devices used for determination of altered protein levels. Hence, Bio sensing methods have great potential as a diagnostic tool for determination of p53 protein. This review describes the screening of most recent and different types of bio sensing approaches, reported for detection of p53. The review also discusses the necessity of biomarker based bio-sensing methods for early diagnosis of cancer. The overall aim of this review is to advance the future analytical approaches of p53 biosensors.

A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.

tRNA-derived fragments (tRFs) have been defined as a novel class of small noncoding RNAs. tRFs have been reported to be deregulated in cancer, but their biologic function remains to be fully understood. We have identified a new tRF (named tRF3E), derived from mature tRNAGlu, that is specifically expressed in healthy mammary glands but not in breast cancer (BC). Consistently, tRF3E levels significantly decrease in the blood of patients with epidermal growth factor receptor 2 (HER2)-positive BC reflecting tumor status (control > early cancer > metastatic cancer). tRF3E down-regulation was recapitulated in Δ16HER2 transgenic mice, representing a BC preclinical model. Pulldown assays, used to search for proteins capable to selectively bind tRF3E, have shown that this tRF specifically interacts with nucleolin (NCL), an RNA-binding protein overexpressed in BC and able to repress the translation of p53 mRNA. The binding properties of NCL-tRF3E complex, predicted in silico and analyzed by EMSA assays, are congruent with a competitive displacement of p53 mRNA by tRF3E, leading to an increased p53 expression and consequently to a modulation of cancer cell growth. Here, we provide evidence that tRF3E plays an important role in the pathogenesis of BC displaying tumor-suppressor functions through a NCL-mediated mechanism.-Falconi, M., Giangrossi, M., Elexpuru Zabaleta, M., Wang, J., Gambini, V., Tilio, M., Bencardino, D., Occhipinti, S., Belletti, B., Laudadio, E., Galeazzi, R., Marchini, C., Amici, A. A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.

[Impaired expression of cell-cycle regulatory proteins in seborrheic keratosis]. / Narushenie ékspressii belkov-reguliatorov kletochnogo tsikla pri seboreinom keratoze.

Seborrheic keratosis (SK) is a benign skin tumor of unknown etiology and pathogenesis. Many details remain unclear despite that there have been a number of studies of cell-cycle abnormalities. AIM: to investigate the expression of the cell-cycle regulatory proteins p53 and p16 and the cell proliferation marker Ki-67 in SK. SUBJECTS AND METHODS: The investigation used intraoperative SK material obtained from 130 patients. Tumors were removed from UV-exposed parts of the body in 63 (48%) patients and from the places that were more often closed in 67 (51.5%). An immunohistochemical (IHC) study was performed using monoclonal antibodies to p53, p16, and Ki-67. RESULTS: A positive reaction with monoclonal antibodies to p53 was recorded in 66 (50.7%) SK samples. In 92.1% of cases, the expression of p53 was found in SK located at the sites that were most exposed to UV radiation (p=0.00001). A positive reaction with monoclonal antibodies to p16 was observed in all SK cases as cytoplasmic staining of more than 50% of the tumor cells: a strong staining in 63 SK samples (overexpression) and a weak staining in 67 SK ones. The level of p16 expression correlated with age (R=0.21; p=0.019) and SK location at the sites exposed to increased insolation (R=0.35; p=0.000038). Overexpressions of p53 and p16 were significantly more commonly recorded in irritated SK. The tumor proliferative activity by the level of Ki-67 expression was low (3.0 to 11.3%). The largest number (8.5±4.8%) of proliferating cells was recorded in irritated SK (p=0.0000001). CONCLUSION: The found disorders in the expression of cell-cycle regulatory proteins in SK are suggestive of tumor suppressor activation and keratinocyte senescence. There may be malignant tumor transformation in irritated SK in terms of the significant increase in the expression of p53, p16 in the presence of high cell proliferative activity.

The Influence of Quadruplex Structure in Proximity to P53 Target Sequences on the Transactivation Potential of P53 Alpha Isoforms.

p53 is one of the most studied tumor suppressor proteins that plays an important role in basic biological processes including cell cycle, DNA damage response, apoptosis, and senescence. The human TP53 gene contains alternative promoters that produce N-terminally truncated proteins and can produce several isoforms due to alternative splicing. p53 function is realized by binding to a specific DNA response element (RE), resulting in the transactivation of target genes. Here, we evaluated the influence of quadruplex DNA structure on the transactivation potential of full-length and N-terminal truncated p53α isoforms in a panel of S. cerevisiae luciferase reporter strains. Our results show that a G-quadruplex prone sequence is not sufficient for transcription activation by p53α isoforms, but the presence of this feature in proximity to a p53 RE leads to a significant reduction of transcriptional activity and changes the dynamics between co-expressed p53α isoforms.

In vitro and in vivo characterization of a novel, highly potent p53-MDM2 inhibitor.

Small molecule inhibitors of the p53-MDM2 protein complex are under intense investigation in clinical trials as anti-cancer agents, including our first generation inhibitor NVP-CGM097. We recently described the rational design of a novel pyrazolopyrrolidinone core as a new lead structure and now we report on the synthesis and optimization of this to provide a highly potent lead compound. This new compound displayed excellent oral efficacy in our preclinical mechanistic in vivo model and marked a significant milestone towards the identification of our second generation clinical candidate NVP-HDM201.