Human P53 Research Articles

5-aza-2'-deoxycytidine induces apoptosis and inhibits tumour growth in vivo of FaDu cells, a specific HPVnegative HNSCC cell line.

Head and neck cancer squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, resulting in over 600,000 new diagnoses annually. Traditionally, HNCC has been related to tobacco and alcohol exposure; however, over the past decade, a growing number of head and neck cancers are attributed to human papillomavirus (HPV) infection. 5-Aza-2’-deoxycytidine (5-AzaD) was demonstrated as an effective chemotherapeutic agent for acute myelogenous leukaemia. Preclinical data revealed that 5-aza inhibits growth and increases cell death of HPV(+) cancer cells. These effects are associated with reduced expression of HPV genes, stabilization of TP53, and activation of TP53-dependent apoptosis. The aim of the present study is to test the effect of 5-AzaD on growth of human squamous cell carcinoma (FaDu), a HPV(-) and p53 mutated cells, in vitro and in vivo. The effect of 5-AzaD on cell viability, cell cycle progression and induction of apoptosis was tested in vitro. The effect of 5-AzaD on tumour growth in vivo was tested using xenograft mice inoculated with FaDu cells. The results indicated that 5-AzaD reduced cell viability and induced apoptosis in FaDu cells in vitro. In vivo studies revealed that 5-AzaD suppresses the growth of tumours in xenograft mice inoculated with FaDu cells through inhibition of proliferation and induction of apoptosis. These findings may emphasis that 5-AzaD is effective in treatment of HPV(-) HNSCC tumours through TP53 independent pathway. Future studies are needed in order to clarify the molecular mechanism of action of 5-AzaD in HPV(-) cancer cells.

Synthesis, structural characterization, density functional theory calculations, and antimicrobial, anticancer, and antimetastatic properties of nanosized heteroleptic complexes of cocaine/TMEDA with d‐block metal ions

AbstractIn the field of transition metal chemistry, the development of transition metal‐based drugs for the treatment of diseases such as cancer or microbial infections with minimization of adverse effects and drug resistance constitutes an active area of research. Herein, eight novel nanosized heteroleptic complexes of cocaine/TMEDA with the formula [M(COC)(TMEDA)Cly(OH2)z]nCl·xH2O (M = Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), and Cd(II); COC = cocaine; TMEDA = N,N,N′,N′‐tetramethylethylenediamine, y = 1–2; z = 0–1; n = 0–1; and x = 0–2) were synthesized and structurally characterized via elemental analysis, molar conductivity, mass spectrometry, and spectroscopic and microscopic techniques. The thermal properties and kinetic thermodynamic parameters of the synthesized complexes were also studied. The geometry and electronic structures were investigated via density functional theory (DFT) calculations. The antiproliferative activity of the complexes on HepG‐2 and MCF‐7 cancer cell lines was quantified via MTT assay. The Fe(III) and Cd(II) complexes exhibited promising cytotoxic activities against the HepG‐2 and MCF‐7 cancer cell lines, respectively, with minimum effect on HFB4 human normal cells. Further molecular mechanistic studies were performed on the Cd(II) complex to inspect its influence on different cancer pathophysiology‐related processes in the MCF‐7 cell line including metastasis, apoptosis, and cellular oxidative stress and on the cellular levels of the human tumor suppressor nuclear proteins p21 and p27. The results revealed that the Cd(II) complex is a promising anticancer agent that acts through several molecular mechanisms with minimum effect on the normal cells and with additional antimetastatic properties. Furthermore, the antibacterial and antifungal activities of the prepared complexes were investigated.

Biocompatibility of 2D silicon nitride: interaction at the nano-bio interface

Determining potential abilities of nanostructures to induce toxicity to biological molecules is still a convoluted challenge in the realm of nanomedicine. Based on the unprecedented achievements of two-dimensional nanomaterials in nearly all areas of applied sciences particularly medicine, we carried out all-atom molecular dynamics simulations to assess the biologically important, yet unmapped issue of biocompatibility of 2D, hexagonal β-Si3N4 nanosheet via investigating its possible cross interactions with both human serum albumin (HSA) and p53 tumor suppressor. Examining the conventional MD indicators in presence and absence of the monolayer revealed that hexagonal Si3N4 nanosheet weakly binds to these two proteins without inducing any important, dramatic change to their secondary structures, revealing accordingly the biological compatibility of the monolayer in case it is released as therapeutics or carriers in vivo. This finding was also broadly supported by the related time-dependent behaviors of the protein-monolayer as well as the protein-water interaction energies.

Molecular Docking of the Cardenolides of Asclepias subulata in the Human p53 Protein Reveals an Interaction in the Cleft of the Y220C Mutant

Background and Objective: The objective of the present study is to use docking and ADME analysis to determine if the cardenolides of Asclepias subulata are potential stabilizing drugs of the p53-Y220C mutant. Materials and Methods: Two different receptors, wild-type p53, and the mutant p53-Y220C, were used for docking. Three independent stochastic series were performed, with 60,000 poses considered, and the 30 best poses were selected. ADME analysis was performed using SwissADME. Results: Docking experiments revealed that corotoxigenin 3-O-glucopyranoside and calotropin interact with the cleft, so they were considered potential stabilizers of the p53-Y220C mutant comparable to the control drug 9H5, which was able to predict a position very similar to that already reported in the crystallographic structure. The ADME predicted that calotropin and desglucouzarin have more favorable pharmacokinetic parameters. Both molecules are predicted to be absorbed from the GIT. Conclusion: Calotropin of A. subulata is predicted to be a potential drug for p53-Y220C, because it binds to the cleft of the mutant and has favorable pharmacokinetic parameters. Corotoxigenin 3- O-glucopyranoside also binds to the Y220C cleft, but had less favorable pharmacokinetic parameters. These results have a future impact since calotropin could be used for the treatment of some types of cancer.

SARS-CoV-2: Understanding the Transcriptional Regulation of ACE2 and TMPRSS2 and the Role of Single Nucleotide Polymorphism (SNP) at Codon 72 of p53 in the Innate Immune Response against Virus Infection.

Human ACE2 and the serine protease TMPRSS2 of novel SARS-CoV-2 are primary entry receptors in host cells. Expression of these genes at the transcriptional level has not been much discussed in detail. The ISRE elements of the ACE2 promoter are a binding site for the ISGF3 complex of the JAK/STAT signaling pathway. TMPRSS2, including IFNβ, STAT1, and STAT2, has the PARP1 binding site near to TSS either up or downstream promoter region. It is well documented that PARP1 regulates gene expression at the transcription level. Therefore, to curb virus infection, both promoting type I IFN signaling to boost innate immunity and prevention of virus entry by inhibiting PARP1, ACE2 or TMPRSS2 are safe options. Most importantly, our aim is to attract the attention of the global scientific community towards the codon 72 Single Nucleotide Polymorphism (SNP) of p53 and its underneath role in the innate immune response against SARS-CoV-2. Here, we discuss codon 72 SNP of human p53′s role in the different innate immune response to restrict virus-mediated mortality rate only in specific parts of the world. In addition, we discuss potential targets and emerging therapies using bioengineered bacteriophage, anti-sense, or CRISPR strategies.

The Changes in the p53 Protein across the Animal Kingdom Point to Its Involvement in Longevity.

Recently, the quest for the mythical fountain of youth has produced extensive research programs that aim to extend the healthy lifespan of humans. Despite advances in our understanding of the aging process, the surprisingly extended lifespan and cancer resistance of some animal species remain unexplained. The p53 protein plays a crucial role in tumor suppression, tissue homeostasis, and aging. Long-lived, cancer-free African elephants have 20 copies of the TP53 gene, including 19 retrogenes (38 alleles), which are partially active, whereas humans possess only one copy of TP53 and have an estimated cancer mortality rate of 11–25%. The mechanism through which p53 contributes to the resolution of the Peto’s paradox in Animalia remains vague. Thus, in this work, we took advantage of the available datasets and inspected the p53 amino acid sequence of phylogenetically related organisms that show variations in their lifespans. We discovered new correlations between specific amino acid deviations in p53 and the lifespans across different animal species. We found that species with extended lifespans have certain characteristic amino acid substitutions in the p53 DNA-binding domain that alter its function, as depicted from the Phenotypic Annotation of p53 Mutations, using the PROVEAN tool or SWISS-MODEL workflow. In addition, the loop 2 region of the human p53 DNA-binding domain was identified as the longest region that was associated with longevity. The 3D model revealed variations in the loop 2 structure in long-lived species when compared with human p53. Our findings show a direct association between specific amino acid residues in p53 protein, changes in p53 functionality, and the extended animal lifespan, and further highlight the importance of p53 protein in aging.

The Δ40p53 isoform inhibits p53-dependent eRNA transcription and enables regulation by signal-specific transcription factors during p53 activation.

The naturally occurring Δ40p53 isoform heterotetramerizes with wild-type p53 (WTp53) to regulate development, aging, and stress responses. How Δ40p53 alters WTp53 function remains enigmatic because their co-expression causes tetramer heterogeneity. We circumvented this issue with a well-tested strategy that expressed Δ40p53:WTp53 as a single transcript, ensuring a 2:2 tetramer stoichiometry. Human MCF10A cell lines expressing Δ40p53:WTp53, WTp53, or WTp53:WTp53 (as controls) from the native TP53 locus were examined with transcriptomics (precision nuclear run-on sequencing [PRO-seq] and RNA sequencing [RNA-seq]), metabolomics, and other methods. Δ40p53:WTp53 was transcriptionally active, and, although phenotypically similar to WTp53 under normal conditions, it failed to induce growth arrest upon Nutlin-induced p53 activation. This occurred via Δ40p53:WTp53-dependent inhibition of enhancer RNA (eRNA) transcription and subsequent failure to induce mRNA biogenesis, despite similar genomic occupancy to WTp53. A different stimulus (5-fluorouracil [5FU]) also showed Δ40p53:WTp53-specific changes in mRNA induction; however, other transcription factors (TFs; e.g., E2F2) could then drive the response, yielding similar outcomes vs. WTp53. Our results establish that Δ40p53 tempers WTp53 function to enable compensatory responses by other stimulus-specific TFs. Such modulation of WTp53 activity may be an essential physiological function for Δ40p53. Moreover, Δ40p53:WTp53 functional distinctions uncovered herein suggest an eRNA requirement for mRNA biogenesis and that human p53 evolved as a tetramer to support eRNA transcription.

Abstract 1351: Targeting mutant p53 protein in spontaneous non-small cell lung cancers in transgenic mice

Abstract Background: Restoration of p53 function in cancer cells is an important approach for cancer therapy because p53 mutations are found in many human cancers, including lung cancers. An anti-mutant p53 agent, p53 reactivation and induction of massive apoptosis (PRIMA-1) has been used to restore the function of the mutant p53 to its wildtype function. Examination of PRIMA-1-induced tumor suppression has indicated the potential role of microRNAs (miRNAs) in the regulation of oncogene or tumor suppressor gene expression. However, the anti-tumor effect of the PRIMA-1 in the treatment of spontaneous lung cancer is still unclear. Methods: We developed a lung tumor model in transgenic mice in which the human mutant p53(273H) is expressed in a lung-specific manner, under the control of the surfactant protein C promoter. These mice developed lung adenocarcinomas after the age of 12 months. We treated SPC-p53-273H lung tumor-bearing mice via intraperitoneal injection (i.p) with PRIMA-1 at a dose of 100 mg/kg in 0.2 ml PBS every other day for three weeks. Lung tumor-bearing mice were identified with a micro-computed tomography (micro-CT) system and pre-and post-treatment tumor volumes were estimated based on the micro-CT image. miRNA expressions were analyzed with the NanoString nCounter Analysis System. Results: Of the 11 treated mice, micro-CT-derived volume changes indicated that five p53-273H mice were responsive to the PRIMA-1 treatment, with an average volume decrease of 93.55 mm3. Four of the treated mice experienced tumor growth post-treatment, with an average volume increase of 74.96 mm3. The remaining two p53-273H mice did not experience a significant change in tumor size. Compared against the control group, the PRIMA-1 treated p53-273H mice showed an overall decrease in tumor size, with an average tumor volume fold change of -2.33. The untreated controls showed a fold change of 8.16, nearly four times that of the treated cohort. Over 500 microRNA were compared between the treated and the control tumors by the NanoString nCounter array analysis. Seven microRNA were identified to have experienced a significant increase (greater than 2 folds) in expression in response to the PRIMA-1 treatment. Fifty-nine miRNAs displayed a significant decrease in expression (less than -2 folds). Conclusion: The results of the study indicated a significant reduction in tumor size in the mice treated with PRIMA-1, based on micro-CT images. Contrary to this finding, an increased average fold change was seen in the control group, indicating an overall average increase in tumor volume. The results indicate the activity of PRIMA-1 in lung tumor-bearing P53 mutant transgenics and suggest the potential feasibility of using PRIMA-1 (APR-246) to treat lung cancer. In addition, microRNA may potentially be involved in the anti-cancer property of PRIMA-1. Further investigation is needed to elucidate the mechanisms in PRIMA-1 induced apoptosis. Citation Format: Shaylyn Grier, Li Gao, Melanie Perez, Wenrui Duan. Targeting mutant p53 protein in spontaneous non-small cell lung cancers in transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1351.

Human papillomavirus DNA and p16 expression in head and neck squamous cell carcinoma in young French patients.

ObjectivesHuman papillomavirus (HPV) is a risk factor for head and neck squamous cell carcinoma (HNSCC), which is currently increasing worldwide. We evaluated the prevalence of HPV DNA and p16 expression in HNSCC patients age <45 years compared with patients aged ≥45 years.MethodsThirty-nine patients aged <45 years who presented at Besançon University Hospital with HNSCC since 2005 were included in this retrospective study. HPV DNA was detected by HPV genotyping and p16 expression was determined by immunohistochemistry using paraffin-embedded tissues. A matched-group of 38 patients aged ≥45 years from Besançon University Hospital was included.ResultsThe overall prevalence of HPV infection was 11.7%. HPV16 was the only genotype detected in 4/39 and 5/38 patients, and p16 was expressed in 6/39 and 4/38 patients aged <45 years and ≥45 years, respectively.ConclusionsHPV-positivity and p16 expression were similar in both age groups. The results suggest that p16 immunohistochemistry may provide a prognosis biomarker for all HNSCCs, not only oropharyngeal cancers, and this should be addressed in large clinical trials.

IMMUNOHISTOCHEMICAL EXPRESSION OF P16 IN CERVICAL AND URINARY BLADDER SQUAMOUS CELL CARCINOMA

Background: p16 is a tumor suppressor protein important in regulating cell cycle. In bladder cancer, p16 mutations are widespread and tend to be more prevalent in low-grade superficial tumors than in higher-grade invasive tumors. Objective: To evaluate expression of p16 in 25 cases of cervical squamous cell carcinoma and 25 cases of urinary bladder squamous cell carcinoma. Patients and methods: This study was carried out in the Histopathology Department of Al-Azhar University Hospitals and from some private laboratories during the period from August 2019 up to January 2020. H & E stained sections were prepared from paraffin blocks for revision of diagnosis. Immunohistochemical analysis was done on 4 micron sections of formalin-fixed paraffin-embedded tissue using the labeled streptavidin-biotin method after antigen retrieval. Antibodies against P16 were utilized on sections from paraffin blocks. CINtec Histology kit includes a two-step antibody method for detection of human p16 antibody. Results: Expression of P16 was positive in 92% of cases of squamous cell carcinoma of the uterine cervix and 60% of cases squamous cell carcinoma of the urinary bladder. In patients with cervical squamous cell carcinoma, there was statistically significant relation between intensity of P16 staining and tumor grading. In patients with squamous cell carcinoma of the urinary bladder, there was a statistically significant relation between intensity of P16 staining and tumor grading and staging. Moderate/strong staining intensity can predict staging 2B among patients with squamous cell carcinoma of the uterine cervix, and negative/weak staining intensity can predict staging 1A, 1B, 2A with sensitivity 52.9% and specificity 37.5% among patients with squamous cell carcinoma of the urinary bladder. Conclusion: p16 immunohistochemical expression alone should not be used as a marker for differentiation between cervical and urinary squamous cell carcinoma.

Stability of p53 oligomers: Tetramerization of p53 impinges on its stability

The p53 protein has been known to exist structurally in three different forms inside the cells. Earlier studies have reported the predominance of the lower oligomeric forms of p53 over its tetrameric form inside the cells, although only the tetrameric p53 contributes to its transcriptional activity. However, it remains unclear the functional relevance of the existence of other p53 oligomers inside the cells. In this study, we characterize the stability and conformational state of tetrameric, dimeric and monomeric p53 that spans both DNA Binding Domain (DBD) and Tetramerization Domain (TD) of human p53 (94–360 amino acid residues). Intriguingly, our studies reveal an unexpected drastic reduction in tetrameric p53 thermal stability in comparison to its dimeric and monomeric form with a higher propensity to aggregate at physiological temperature. Our EMSA study suggests that tetrameric p53, not their lower oligomeric counterpart, exhibit rapid loss of binding to their consensus DNA elements at the physiological temperature. This detrimental effect of destabilization is imparted due to the tetramerization of p53 that drives the DBDs to misfold at a faster pace when compared to its lower oligomeric form. This crosstalk between DBDs is achieved when it exists as a tetramer but not as dimer or monomer. Our findings throw light on the plausible reason for the predominant existence of p53 in dimer and monomer forms inside the cells with a lesser population of tetramer form. Therefore, the transient disruption of tetramerization between TDs could be a potential cue for the stabilization of p53 inside the cells.

Implementing a method for engineering multivalency to substantially enhance binding of clinical trial anti-SARS-CoV-2 antibodies to wildtype spike and variants of concern proteins

Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 disease. Therapeutic antibodies are being developed that interact with the viral spike proteins to limit viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed quads, that retain efficient binding to the SARS-CoV-2 spike protein, show up to two orders of magnitude enhancement in neutralization of pseudovirus infection and retain potent interaction with virus variant of concern spike proteins. The tetramerization method is simple, general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation.

Human Papillomavirus and p16 Expression in the Female Genital Tract and Its Value in Diagnosis

Human Papillomavirus and p16 Expression in the Female Genital Tract and Its Value in Diagnosis

A tetravalent single-chain variable fragment antibody for the detection of staphylococcal enterotoxin A.

Staphylococcal enterotoxin A (SEA) synthesized by Staphylococcus aureus is a foodborne and heat-stable toxin, which is a great threat to human health (Pexaraet al., 2010). Highly sensitive antibodies are a key factor in the immunological detection of SEA, which is one of the most effective ways to detect SEA because of its accuracy, agility, and efficiency (Nouri et al., 2018). In this study, we constructed a tetravalent anti-SEA antibody gene by linking the tetramerization domain of human p53 to the C-terminus of the anti-SEA single-chain variable fragment (scFv), which was then transformed into Escherichia coli BL21 (DE3) for the production of a SEA-specific tetravalent antibody. Successful expression of the tetravalent antibody was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. An indirect non-competitive enzyme-linked immunosorbent assay (ELISA) revealed that the tetravalent antibody exhibited SEA-specific binding activity. A sandwich ELISA demonstrated that compared to the scFv monomer, the tetravalent antibody was more sensitive in detecting SEA. Molecular docking analysis revealed that the SEA interacted with the scFv mainly on the opposite side of the residue linked to p53. Thus, this study indicated that genetically engineered tetramerization is a potential way to improve the sensitivity of SEA-specific scFv.

Loss-of-function of p53 isoform \u0394113p53 accelerates brain aging in zebrafish

Reactive oxygen species (ROS) stress has been demonstrated as potentially critical for induction and maintenance of cellular senescence, and been considered as a contributing factor in aging and in various neurological disorders including Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). In response to low-level ROS stress, the expression of Δ133p53, a human p53 isoform, is upregulated to promote cell survival and protect cells from senescence by enhancing the expression of antioxidant genes. In normal conditions, the basal expression of Δ133p53 prevents human fibroblasts, T lymphocytes, and astrocytes from replicative senescence. It has been also found that brain tissues from AD and ALS patients showed decreased Δ133p53 expression. However, it is uncharacterized if Δ133p53 plays a role in brain aging. Here, we report that zebrafish Δ113p53, an ortholog of human Δ133p53, mainly expressed in some of the radial glial cells along the telencephalon ventricular zone in a full-length p53-dependent manner. EDU-labeling and cell lineage tracing showed that Δ113p53-positive cells underwent cell proliferation to contribute to the neuron renewal process. Importantly, Δ113p53M/M mutant telencephalon possessed less proliferation cells and more senescent cells compared to wild-type (WT) zebrafish telencephalon since 9-months old, which was associated with decreased antioxidant genes expression and increased level of ROS in the mutant telencephalon. More interestingly, unlike the mutant fish at 5-months old with cognition ability, Δ113p53M/M zebrafish, but not WT zebrafish, lost their learning and memory ability at 19-months old. The results demonstrate that Δ113p53 protects the brain from aging by its antioxidant function. Our finding provides evidence at the organism level to show that depletion of Δ113p53/Δ133p53 may result in long-term ROS stress, and finally lead to age-related diseases, such as AD and ALS in humans.

P53 and p16 expression profiles in vulvar cancer: a translational analysis by the Arbeitsgemeinschaft Gynäkologische Onkologie Chemo and Radiotherapy in Epithelial Vulvar Cancer study group

There are 2 known pathways for tumorigenesis of vulvar squamous cell carcinoma-a human papillomavirus-dependent pathway characterized by p16 overexpression and a human papillomavirus-independent pathway linked to lichen sclerosus, characterized by TP53 mutation. A correlation of human papillomavirus dependency with a favorable prognosis has been proposed. The objective of the study was to further understand the role of human papillomavirus and p53 status in vulvar squamous cell carcinoma and characterize its clinical relevance. The Arbeitsgemeinschaft Gynaecological Oncology Chemo and Radiotherapy in Epithelial Vulvar Cancer-1 study is a retrospective cohort study of 1618 patients with primary vulvar squamous cell carcinoma Fédération Internationale de Gynécologie et d'Obstétrique stage ≥1B treated at 29 gynecologic cancer centers in Germany between 1998 and 2008. For this translational substudy, formalin-fixed paraffin-embedded tissue was collected. A tissue microarray was constructed (n=652 samples); p16 and p53 expression was determined by immunohistochemistry. Human papillomavirus status and subtype were analyzed by polymerase chain reaction. p16 immunohistochemistry was positive in 166 of 550 tumors (30.2%); p53 staining in 187 of 597 tumors (31.3%). Only tumors with available information regarding p16 and p53 immunohistochemistry and without p53 silent expression pattern were further analyzed (n=411); 3 groups were defined: p53+ (n=163), p16+/p53- (n=132), and p16-/p53- (n=116). Human papillomavirus DNA was detected in 85.6% of p16+/p53- tumors; human papillomavirus-16 was the most common subtype (86.3%). Patients with p16+ tumors were younger (64 vs 72 years for p53+, respectively, 69 years for p16-/p53- tumors; P<.0001) and showed lower rates of lymph-node involvement (28.0% vs 42.3% for p53+, respectively, 30.2% for p16-/p53- tumors; P=.050). Notably, 2-year-disease-free and overall survival rates were significantly different among the groups: disease-free survival, 47.1% (p53+), 60.2% (p16-/p53-), and 63.9% (p16+/p53-) (P<.001); overall survival, 70.4% (p53+), 75.4% (p16-/p53-), and 82.5% (p16+/p53-) (P=.002). In multivariate analysis, the p16+/p53- phenotype showed a consistently improved prognosis compared with the other groups (hazard ratio, 0.66; 95% confidence interval, 0.44-0.99; P=.042). p16 overexpression is associated with an improved prognosis whereas p53 positivity is linked to an adverse outcome. Our data support the hypothesis of a clinically relevant third subgroup of vulvar squamous cell carcinoma with a p53-/p16- phenotype showing an intermediate prognosis that needs to be further characterized.

Urea Denaturation, Zinc Binding, and DNA Binding Assays of Mutant p53 DNA-binding Domains and Full-length Proteins.

In the cell, the thermodynamic stability of a protein - and hence its biological activity - can change dramatically as a result of perturbations in its amino acid sequence and the concentration of stabilizing ligands. This interplay is particularly evident in zinc-binding transcription factors such as the p53 tumor suppressor, whose DNA-binding activity can critically depend on levels of intracellular zinc as well as point mutations that alter either metal binding or folding stability. Separate protocols exist for determining a protein's metal affinity and its folding free energy. These properties, however, are intimately connected, and a technique is needed to integrate these measurements. Our protocols employ common non-fluorescent and fluorescent zinc chelators to control and report on free Zn2+ concentration, respectively, combined with biophysical assays of full-length human p53 and its DNA-binding domain. Fitting the data to equations that contain stability and metal-binding terms results in a more complete picture of how metal-dependent proteins can lose and gain DNA-binding function in a range of physiological conditions. Graphic abstract: Figure 1.Raising intracellular zinc can restore tumor-suppressing function to p53 that has been unfolded by missense mutation or cellular conditions.

A cell permeable bimane-constrained PCNA-interacting peptide.

The human sliding clamp protein known as proliferating cell nuclear antigen (PCNA) orchestrates DNA-replication and -repair and as such is an ideal therapeutic target for proliferative diseases, including cancer. Peptides derived from the human p21 protein bind PCNA with high affinity via a 310-helical binding conformation and are known to shut down DNA-replication. Here, we present studies on short analogues of p21 peptides (143–151) conformationally constrained with a covalent linker between i, i + 4 separated cysteine residues at positions 145 and 149 to access peptidomimetics that target PCNA. The resulting macrocycles bind PCNA with KD values ranging from 570 nM to 3.86 μM, with the bimane-constrained peptide 7 proving the most potent. Subsequent X-ray crystallography and computational modelling studies of the macrocyclic peptides bound to PCNA indicated only the high-affinity peptide 7 adopted the classical 310-helical binding conformation. This suggests the 310-helical conformation is critical to high affinity PCNA binding, however NMR secondary shift analysis of peptide 7 revealed this secondary structure was not well-defined in solution. Peptide 7 is cell permeable and localised to the cell cytosol of breast cancer cells (MDA-MB-468), revealed by confocal microscopy showing blue fluorescence of the bimane linker. The inherent fluorescence of the bimane moiety present in peptide 7 allowed it to be directly imaged in the cell uptake assay, without attachment of an auxiliary fluorescent tag. This highlights a significant benefit of using a bimane constraint to access conformationally constrained macrocyclic peptides. This study identifies a small peptidomimetic that binds PCNA with higher affinity than previous reported p21 macrocycles, and is cell permeable, providing a significant advance toward development of a PCNA inhibitor for therapeutic applications.

Transplantation of human corneal limbal epithelial cell sheet harvested on synthesized carboxymethyl cellulose and dopamine in a limbal stem cell deficiency.

This study aimed to evaluate the efficacy and safety of transplantation with human corneal limbal epithelial (HCLE) cell sheets cultured on carboxymethyl cellulose (CMC)-dopamine (DA)-coated substrates and harvested via enzymatic digestion of CMC with cellulase in a rabbit animal model of limbal stem cell deficiency (LSCD). Synthesized CMC-DA was pretreated onto the surface of culture plates. Then, HCLE cells were cultured on precoated CMC-DA and HCLE cell sheets were harvested using cellulase-containing cell culture medium. HCLE cell sheets were evaluated using a live/dead assay, histological examination, and immunofluorescence staining. For in vivo assessment, HCLE cell sheets were transplanted in a rabbit model of LSCD for 2 weeks to determine the effectiveness of the repair. Primary culture of HCLE cells stained positively for p63, cytokeratin (CK)15, and CK12. HCLE cell sheets were generated with a well-preserved morphology and transparency ranging in size from 15 to 19 mm after cellulase-assisted cell sheet generation. HCLE cell sheets uniformly stained positively for human mitochondria, p63, CK15, CK12, CK3/2p, and zonula occludens (ZO)-1. HCLE cell sheet transplantation in a rabbit model of LSCD improved the corneal opacity and neovascularization scores. Transplanted HCLE cell sheets stained positively for p63 and CK12. Transplantation of HCLE cell sheets harvested on CMC-DA coating combined with cellulase is a safe and efficient procedure for corneal epithelial regeneration in a rabbit model of LSCD. This system could enable a promising strategy to regenerate corneal epithelium by transplantation in ocular surface disorders.

THER-08. SGT53 – A NOVEL p53 NANOMEDICINE INDUCES SIGNIFICANT RESPONSES IN CHILDREN WITH RECURRENT MEDULLOBLASTOMA AND CHOROID PLEXUS CARCINOMA: A REPORT OF TWO CASES

BACKGROUNDAbnormal p53 function commonly defines high-risk CNS tumors, but functional restoration has eluded investigators. SGT-53 is a targeted nanomedicine encapsulating a plasmid DNA encoding wild-type human p53 with a transferrin receptor-targeting scFv on the nanocomplex surface resulting in efficient delivery across the BBB and robust tumor binding/uptake. Data generated by collaborators demonstrated synergy with irradiation and chemotherapy. REPORT: Two children with recurrent CNS malignancies have been treated with SGT-53, each receiving greater than 50 infusions in combination with irradiation and chemotherapy with no grade 3/4 AEs positively attributed to SGT-53. The first was an 11yo male with recurrent disseminated p53+ SHH medulloblastoma, with bulky intracranial/thoracolumbar disease. He received irradiation followed by biweekly doses of SGT-53 and temozolomide, bevacizumab and irinotecan given one week out of four. This patient exhibited a complete response of all disease on his first follow-up scan 8 weeks after therapy initiation and remained in remission for 8 months. The second patient was a 3yo male with disseminated recurrent choroid plexus carcinoma. He received CSI with SGT-53, followed by SGT-53, temozolomide and irinotecan as described above, again with no related grade 3/4 AEs. He experienced a partial response to all sites of disease and completed therapy after six months, progressing 7.8 months after initiating treatment.CONCLUSIONSSGT-53 was well tolerated in two heavily-pretreated patients despite aggressive combinatorial strategies. The majority of the AEs experienced were mild and manageable. Each patient had significant responses, suggesting that SGT-53 should be evaluated in a clinical trial for similar patients.