RESUMO
Pregnancy is characterized by a delicate immune balance; therefore, infectious diseases might increase the risk of adverse pregnancy outcomes (APOs). Here, we hypothesize that pyroptosis, a unique cell death pathway mediated by the NLRP3 inflammasome, could link SARS-CoV-2 infection, inflammation, and APOs. Two blood samples were collected from 231 pregnant women at 11-13 weeks of gestation and in the perinatal period. At each time point, SARS-CoV-2 antibodies and neutralizing antibody titers were measured by ELISA and microneutralization (MN) assays, respectively. Plasmatic NLRP3 was determined by ELISA. Fourteen miRNAs selected for their role in inflammation and/or pregnancy were quantified by qPCR and further investigated by miRNA-gene target analysis. NLRP3 levels were positively associated with nine circulating miRNAs, of which miR-195-5p was increased only in MN+ women (p-value = 0.017). Pre-eclampsia was associated with a decrease in miR-106a-5p (p-value = 0.050). miR-106a-5p (p-value = 0.026) and miR-210-3p (p-value = 0.035) were increased in women with gestational diabetes. Women giving birth to small for gestational age babies had lower miR-106a-5p and miR-21-5p (p-values = 0.001 and 0.036, respectively), and higher miR-155-5p levels (p-value = 0.008). We also observed that neutralizing antibodies and NLRP3 concentrations could affect the association between APOs and miRNAs. Our findings suggest for the first time a possible link between COVID-19, NLRP3-mediated pyroptosis, inflammation, and APOs. Circulating miRNAs might be suitable candidates to gain a comprehensive view of this complex interplay.
Assuntos
COVID-19 , MicroRNA Circulante , MicroRNAs , Humanos , Gravidez , Feminino , Resultado da Gravidez , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Piroptose , SARS-CoV-2/metabolismo , MicroRNAs/metabolismo , InflamaçãoRESUMO
Chronic lymphocytic leukemia (CLL) is a hematological disorder with complex clinical and biological behavior. TP53 mutational status and cytogenetic assessment of the deletion of the corresponding locus (17p13.1) are considered the most relevant biomarkers associated with pharmaco-predictive response, chemo-refractoriness, and worse prognosis in CLL patients. The implementation of Next Generation Sequencing (NGS) methodologies in the clinical laboratory allows for comprehensively analyzing the TP53 gene and detecting mutations with allele frequencies ≤10%, that is, "subclonal mutations". We retrospectively studied TP53 gene mutational status by NGS in 220 samples from 171 CLL patients. TP53 mutations were found in 60/220 (27.3%) samples and 47/171 (27.5%) patients. Interestingly, subclonal mutations could be detected in 31/60 samples (51.7%) corresponding to 25 patients (25/47, 53.2%). We identified 44 distinct subclonal TP53 mutations clustered in the central DNA-binding domain of p53 protein (exons 5-8, codons 133-286). Missense mutations were predominant (>80%), whereas indels, nonsense, and splice site variants were less represented. All subclonal TP53 variants but one [p.(Pro191fs)] were already described in NCI and/or Seshat databases as "damaging" and/or "probably damaging" mutations (38/44, 86% and 6/44, 14%, respectively). Longitudinal samples were available for 37 patients. Almost half of them displayed at least one TP53 mutant subclone, which could be alone (4/16, 25%) or concomitant with other TP53 mutant clonal ones (12/16, 75%); different patterns of mutational dynamics overtimes were documented. In conclusion, utilization of NGS in our "real-life" cohort of CLL patients demonstrated an elevated frequency of subclonal TP53 mutations. This finding indicates the need for precisely identifying these mutations during disease since the clones carrying them may become predominant and be responsible for therapy failures.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Leucemia Linfocítica Crônica de Células B , Humanos , Proteína Supressora de Tumor p53/genética , Leucemia Linfocítica Crônica de Células B/genética , Estudos RetrospectivosRESUMO
The formation of a tetrameric assembly is essential for the ability of the tumor suppressor protein p53 to act as a transcription factor. Such a quaternary conformation is driven by a specific tetramerization domain, separated from the central DNA-binding domain by a flexible linker. Despite the distance, functional crosstalk between the two domains has been reported. This phenomenon can explain the pathogenicity of some inherited or somatically acquired mutations in the tetramerization domain, including the widespread R337H missense mutation present in the population in south Brazil. In this work, we combined computational predictions through extended all-atom molecular dynamics simulations with functional assays in a genetically defined yeast-based model system to reveal structural features of p53 tetramerization domains and their transactivation capacity and specificity. In addition to the germline and cancer-associated R337H and R337C, other rationally designed missense mutations targeting a significant salt-bridge interaction that stabilizes the p53 tetramerization domain were studied (i.e., R337D, D352R, and the double-mutation R337D plus D352R). The simulations revealed a destabilizing effect of the pathogenic mutations within the p53 tetramerization domain and highlighted the importance of electrostatic interactions between residues 337 and 352. The transactivation assay, performed in yeast by tuning the expression of wild-type and mutant p53 proteins, revealed that p53 tetramerization mutations could decrease the transactivation potential and alter transactivation specificity, in particular by better tolerating negative features in weak DNA-binding sites. These results establish the effect of naturally occurring variations at positions 337 and 352 on p53's conformational stability and function.
Assuntos
Saccharomyces cerevisiae , Proteína Supressora de Tumor p53 , DNA , Proteínas Mutantes/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Anicteric gallbladder rupture has been rarely described in veterinary medicine, and, generally, it has been related to gallbladder wall necrosis secondary to gallbladder mucocele. A 5 yr old, male, neutered Labrador retriever presented for acute onset anorexia, lethargy, and vomiting. Cholecystitis was diagnosed based on the ultrasonographic findings and bactibilia, and, consequently, medical treatment was established. Despite improvement of the patient, a focal ultrasound of the hepatobiliary tract was performed 72 hr after admission for reassessment, revealing gallbladder wall thickening and abdominal effusion. Intracellular bacteria were present in nondegenerated neutrophils, and the effusion was categorized as septic exudate, compatible with septic peritonitis. Exploratory laparotomy confirmed an anicteric gallbladder rupture potentially secondary to cholecystitis and/or previous cholecystocentesis. The patient was not icteric the day of the surgery, serum bilirubin was within normal limits, abdominal fluid bilirubin concentration was below that of serum, and no bile pigment was detected; however, bile acids were significantly higher in the abdominal effusion compared with the serum concentration. This case describes an anicteric gallbladder rupture in a dog with concomitant cholecystitis and raises the question about the sensitivity of bile acid evaluation as a tool for diagnosis of gallbladder rupture and bile peritonitis in dogs.
Assuntos
Colecistite , Doenças do Cão , Doenças da Vesícula Biliar , Peritonite , Animais , Ácidos e Sais Biliares , Bilirrubina , Colecistite/complicações , Colecistite/diagnóstico , Colecistite/cirurgia , Colecistite/veterinária , Doenças do Cão/diagnóstico , Doenças do Cão/cirurgia , Cães , Exsudatos e Transudatos , Doenças da Vesícula Biliar/complicações , Doenças da Vesícula Biliar/cirurgia , Doenças da Vesícula Biliar/veterinária , Masculino , Peritonite/complicações , Peritonite/diagnóstico , Peritonite/veterinária , Ruptura/veterináriaRESUMO
OBJECTIVES: To compare the capacity of ibrutinib (IB) and idelalisib-rituximab (IDELA-R) of prolonging overall survival (OS) as in CLL patients, previously treated with chemotherapy only. METHODS: A real-life cohort of 675 cases has been identified and investigated in the database of the groups participating in the study. RESULTS: At an unadjusted univariate analysis, a significant death risk reduction was observed favoring IB (IDELA-R vs IB HR = 0.5, 95% CI = 0.36-0.71) although with some limitations due to the non-randomized and retrospective nature of the study and to the lower number of patients in the IDELA-R group (112 cases) related to the current prescribing practice. To overcome the potential problem of confounding by indication, we adjusted the association between the type of therapy and mortality for all variables significantly associated with OS at Cox univariate analysis. Furthermore, those variables, differently distributed between the two study groups, were introduced into the multivariate Cox model to improve the effectiveness of the analysis. By introducing all these variables into the multiple Cox regression model, we confirmed the protective effect of IB vs IDELA-R (HR = 0.67, 95% CI = 0.45-0.98, P = .04) independent of potential confounders. CONCLUSIONS: Although our analysis presents some constraints, that is, the unavailability of additional potential confounders, and the retrospective nature of the study, this observation may be of help for the daily clinical practice, particularly in the absence of randomized trials comparing the two schedules.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Adenina/administração & dosagem , Adenina/análogos & derivados , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores Tumorais , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Imunoglobulinas/genética , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/etiologia , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Mutação , Piperidinas/administração & dosagem , Modelos de Riscos Proporcionais , Purinas/administração & dosagem , Quinazolinonas/administração & dosagem , Recidiva , Retratamento , Rituximab/administração & dosagem , Resultado do TratamentoRESUMO
Tumor grading is a method to quantify the putative clinical aggressiveness of a neoplasm based on specific histological features. A good grading system should be simple, easy to use, reproducible, and accurately segregate tumors into those with low versus high risk. The aim of this review is to summarize the histological and, when available, cytological grading systems applied in veterinary pathology, providing information regarding their prognostic impact, reproducibility, usefulness, and shortcomings. Most of the grading schemes used in veterinary medicine are developed for common tumor entities. Grading systems exist for soft tissue sarcoma, osteosarcoma, multilobular tumor of bone, mast cell tumor, lymphoma, mammary carcinoma, pulmonary carcinoma, urothelial carcinoma, renal cell carcinoma, prostatic carcinoma, and central nervous system tumors. The prognostic relevance of many grading schemes has been demonstrated, but for some tumor types the usefulness of grading remains controversial. Furthermore, validation studies are available only for a minority of the grading systems. Contrasting data on the prognostic power of some grading systems, lack of detailed instructions in the materials and methods in some studies, and lack of data on reproducibility and validation studies are discussed for the relevant grading systems. Awareness of the limitations of grading is necessary for pathologists and oncologists to use these systems appropriately and to drive initiatives for their improvement.
Assuntos
Carcinoma de Células de Transição , Neoplasias Renais , Neoplasias da Bexiga Urinária , Animais , Carcinoma de Células de Transição/veterinária , Neoplasias Renais/veterinária , Gradação de Tumores , Prognóstico , Reprodutibilidade dos Testes , Neoplasias da Bexiga Urinária/veterináriaRESUMO
Mutant p53 (mutp53) proteins are frequently present at higher levels than the wild-type (wt) protein in tumors, and some of them can acquire oncogenic properties. Consistently, knockdown of mutp53 protein in human cancer cell lines leads to reduced cell proliferation and invasion as well as to an increased sensitivity to some anticancer drugs. Therefore, the exploitation of cellular pathways and/or molecules that promote mutp53 degradation may have a therapeutic interest. Recently, autophagy is emerging as an important pathway involved in the stability of mutp53. In this paper, we explored the autophagic potential of gambogic acid (GA), a molecule that stimulates the degradation of mutp53 and increases the sensitivity of cancer cells to chemotherapeutic agents. We demonstrated that GA may induce mutp53 degradation through autophagy in cancer cells expressing the p53-R280K (MDA-MB-231) and the p53-S241F (DLD1) proteins. The inhibition of autophagy with bafilomycin A1 or chloroquine counteracted mutp53 degradation by GA. However, the autophagy induction and mutp53 degradation affected cell survival and proliferation only at low GA concentrations. At higher GA concentrations, when cells undergo massive apoptosis, autophagy is no longer detectable by immuno-fluorescence analysis. We concluded that autophagy is a relevant pathway for mutp53 degradation in cancer cells but it contributes only partially to GA-induced cell death, in a time and dose-dependent manner.
Assuntos
Autofagia/efeitos dos fármacos , Mutação , Estabilidade Proteica/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Xantonas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Proteína Supressora de Tumor p53/genéticaRESUMO
Vaginal and vulvar tumors are uncommon in dogs. Knowledge of canine primary clitoral neoplasia is restricted to a few case reports, and only carcinomas have been reported. Cytologic and histologic features reported in the literature seem to overlap with those of canine apocrine gland anal sac adenocarcinoma (AGASA). Clinical features also recall those of canine AGASA, such as locoregional metastases and hypercalcemia of malignancy (HM). In this study, 6 cases of primary canine clitoral carcinomas (CCCs), with and without HM, were investigated by means of cytology, histopathology, electron microscopy, and immunohistochemistry for neuroendocrine markers including chromogranin A (CGA), synaptophysin (SYN), neuron-specific enolase (NSE), and S-100. In all 6 tumors, cytologic findings were consistent with malignant epithelial neoplasia of apocrine gland origin. The tumors examined were classified into 3 different histological patterns representing different degrees of differentiation: tubular, solid, and rosette type. Both CGA and SYN were mildly expressed in 2 of 6 tumors, while NSE was consistently expressed in all 6 cases. None of the tumors were S-100 positive. Transmission electron microscopy revealed electron-dense cytoplasmic granules compatible with neuroendocrine granules in all 6 cases. CCCs presented clinicopathologic features resembling AGASAs with neuroendocrine characteristics, and 2 of 6 neoplasms were considered as carcinomas with neuroendocrine differentiation and were positive for 3 neuroendocrine markers. CCCs can often present with HM, and long-term outcome is likely poor. Our study concludes that CCC seems to be a rare tumor, but it might be underestimated because of the overlapping features with AGASA. Further studies should aim to define the true incidence of this disease.
Assuntos
Adenocarcinoma/patologia , Biomarcadores Tumorais/análise , Carcinoma/veterinária , Doenças do Cão/patologia , Hipercalcemia/veterinária , Síndromes Paraneoplásicas/veterinária , Adenocarcinoma/diagnóstico , Adenocarcinoma/cirurgia , Adenocarcinoma/ultraestrutura , Animais , Carcinoma/diagnóstico , Carcinoma/patologia , Carcinoma/ultraestrutura , Cromogranina A/análise , Clitóris/patologia , Doenças do Cão/diagnóstico , Doenças do Cão/cirurgia , Cães , Feminino , Hipercalcemia/diagnóstico , Hipercalcemia/patologia , Imuno-Histoquímica/veterinária , Síndromes Paraneoplásicas/diagnóstico , Síndromes Paraneoplásicas/patologia , Sinaptofisina/análise , Vulva/patologiaRESUMO
The observation that human transcription factors (TFs) can function when expressed in yeast cells has stimulated the development of various functional assays to investigate (i) the role of binding site sequences (herein referred to as response elements, REs) in transactivation specificity, (ii) the impact of polymorphic nucleotide variants on transactivation potential, (iii) the functional consequences of mutations in TFs and (iv) the impact of cofactors or small molecules. These approaches have found applications in basic as well as applied research, including the identification and the characterisation of mutant TF alleles from clinical samples. The ease of genome editing of yeast cells and the availability of regulated systems for ectopic protein expression enabled the development of quantitative reporter systems, integrated at a chosen chromosomal locus in isogenic yeast strains that differ only at the level of a specific RE targeted by a TF or for the expression of distinct TF alleles. In many cases, these assays were proven predictive of results in higher eukaryotes. The potential to work in small volume formats and the availability of yeast strains with modified chemical uptake have enhanced the scalability of these approaches. Next to well-established one-, two-, three-hybrid assays, the functional assays with non-chimeric human TFs enrich the palette of opportunities for functional characterisation. We review â¼25 years of research on human sequence-specific TFs expressed in yeast, with an emphasis on the P53 and NF-кB family of proteins, highlighting outcomes, advantages, challenges and limitations of these heterologous assays.
Assuntos
NF-kappa B/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Transgenes , Proteína Supressora de Tumor p53/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Expressão Gênica , Marcação de Genes/métodos , Genética Microbiana/métodos , Humanos , NF-kappa B/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Proteína Supressora de Tumor p53/genéticaAssuntos
Adenina/análogos & derivados , Cloridrato de Bendamustina/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Piperidinas/uso terapêutico , Rituximab/uso terapêutico , Adenina/farmacologia , Adenina/uso terapêutico , Idoso , Cloridrato de Bendamustina/farmacologia , Humanos , Piperidinas/farmacologia , Intervalo Livre de Progressão , Rituximab/farmacologiaRESUMO
In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions.
Assuntos
Autofagia , Saccharomyces cerevisiae/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetatos/farmacologia , Actinas/genética , Actinas/metabolismo , Proteínas de Ciclo Celular , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Imidazóis/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Piperazinas/farmacologia , Ligação Proteica , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pirazóis/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
A novel resource centre for TP53 mutations and mutants has been developed (http://p53.fr). TP53 gene dysfunction can be found in the majority of human cancer types. The potential use of TP53 mutation as a biomarker for clinical studies or exposome analysis has led to the publication of thousands of reports describing the TP53 gene status in >10,000 tumours. The UMD TP53 mutation database was created in 1990 and has been regularly updated. The 2012 release of the database has been carefully curated, and all suspicious reports have been eliminated. It is available either as a flat file that can be easily manipulated or as novel multi-platform analytical software that has been designed to analyse various aspects of TP53 mutations. Several tools to ascertain TP53 mutations are also available for download. We have developed TP53MULTLoad, a manually curated database providing comprehensive details on the properties of 2549 missense TP53 mutants. More than 100,000 entries have been arranged in 39 different activity fields, such as change of transactivation on various promoters, apoptosis or growth arrest. For several hot spot mutants, multiple gain of function activities are also included. The database can be easily browsed via a graphical user interface.
Assuntos
Bases de Dados de Ácidos Nucleicos , Genes p53 , Proteína Supressora de Tumor p53/genética , Humanos , Internet , Mutação , Neoplasias/genéticaRESUMO
Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein-DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas Nucleares/química , Elementos de Resposta , Transativadores/química , Ativação Transcricional , Proteína Supressora de Tumor p53/química , Proteínas Supressoras de Tumor/química , Alelos , Sequência de Bases , Linhagem Celular Tumoral , Sequência Consenso , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Fenilalanina/química , Estrutura Terciária de Proteína , Purinas/análise , Pirimidinas/análise , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Transativadores/genética , Transativadores/metabolismo , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The transcription factor p73 triggers developmental pathways and overlaps stress-induced p53 transcriptional pathways. How p53-family response elements determine and regulate transcriptional specificity remains an unsolved problem. In this work, we have determined the first crystal structures of p73 DNA-binding domain tetramer bound to response elements with spacers of different length. The structure and function of the adaptable tetramer are determined by the distance between two half-sites. The structures with zero and one base-pair spacers show compact p73 DNA-binding domain tetramers with large tetramerization interfaces; a two base-pair spacer results in DNA unwinding and a smaller tetramerization interface, whereas a four base-pair spacer hinders tetramerization. Functionally, p73 is more sensitive to spacer length than p53, with one base-pair spacer reducing 90% of transactivation activity and longer spacers reducing transactivation to basal levels. Our results establish the quaternary structure of the p73 DNA-binding domain required as a scaffold to promote transactivation.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Proteínas Nucleares/química , Multimerização Proteica , Estrutura Terciária de Proteína , Ativação Transcricional , Proteínas Supressoras de Tumor/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Quaternária de Proteína , Elementos de Resposta/genética , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Loss-of-function, partial-function, altered-function, dominant-negative, temperature sensitive, interfering, contact, structural, unfolded, misfolded, dimeric, monomeric, non-cooperative, unstable, supertrans, superstable, intragenic suppressor. TP53 mutants are many, more than 2,000 in fact, and they can be very diverse. Sporadic; germline; gain-of-function (GoF); oncogenic; rebel-angel; yin and yang; prion-like; metastasis-inducer; mediator of chemo-resistance; modifier of stemness. TP53 mutants can impact important cancer clinical variables, in multiple, often subtle ways, as revealed by cell-based assays as well as animal models. Here, we review studies investigating TP53 mutants for their effect on sequence-specific transactivation function, and especially recent findings on how TP53 mutants can exhibit GoF properties. We also review reports on TP53 mutants' impact on cancer cell transcriptomes and studies with Li-Fraumeni patients trying to classify and predict phenotypes in relation to experimentally determined transcription fingerprints. Finally, we provide an example of the complexity of correlating TP53 mutant functionality to clinical variables in sporadic cancer patients. Conflicting results and limitations of experimental approaches notwithstanding, the study of TP53 mutants has provided a rich body of knowledge, mostly available in the public domain and accessible through databases, which is beginning to impact cancer intervention strategies.
Assuntos
Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Síndrome de Li-Fraumeni/genética , Proteína Supressora de Tumor p53/genética , Bases de Dados Genéticas , Genótipo , Humanos , Síndrome de Li-Fraumeni/patologia , Fenótipo , Proteína Supressora de Tumor p53/metabolismoRESUMO
N3-methyladenine (3-mA), generated by the reaction of methylating agents with DNA, is considered a highly toxic but weakly mutagenic lesion. However, due to its intrinsic instability, some of the biological effects of the adduct can result from the formation of the corresponding depurination product [an apurinic (AP)-site]. Previously, we exploited Me-lex, i.e. {1-methyl-4-[1-methyl-4-(3-methoxysulfonylpropanamido)pyrrole-2-carboxamido]-pyrrole-2 carboxamido}propane, a minor groove equilibrium binder with selectivity for A/T rich sequences that efficiently reacts with DNA to afford 3-mA as the dominant product, to probe the biology of this lesion. Using human p53 cDNA as a target in a yeast system, a weak increase in mutagenicity was observed in the absence of Mag1 (3-methyladenine-DNA glycosylase 1, mag1), the enzyme devoted to remove 3-mA from DNA. Moreover, a significant increase in mutagenicity occurred in the absence of the enzymes involved in the repair of AP-sites (AP endonucleases 1 and 2, apn1apn2). Since methyl methanesulfonate (MMS) has been extensively used to explore the biological effects of 3-mA, even though it produces 3-mA in low relative yield, we compared the toxicity and mutagenicity induced by MMS and Me-lex in yeast. A mutagenesis reporter plasmid was damaged in vitro by MMS and then transformed into wild-type and Translesion Synthesis (TLS) Polζ (REV3) and Polη (RAD30) deficient strains. Furthermore, a mag1rad30 double mutant strain was constructed and transformed with the DNA plasmid damaged in vitro by Me-lex. The results confirm the important role of Polζ in the mutagenic bypass of MMS and Me-lex induced lesions, with Polη contributing only towards the bypass of Me-lex induced lesions, mainly in an error-free way. Previous and present results point towards the involvement of AP-sites, derived from the depurination of 3-mA, in the observed toxicity and mutagenicity.
Assuntos
Adenina/análogos & derivados , Metanossulfonato de Metila/toxicidade , Mutagênicos/toxicidade , Netropsina/análogos & derivados , Adenina/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Humanos , Netropsina/toxicidade , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
The effect of mutations in the P53 family of transcription factors on their biological functions, including partial or complete loss of transcriptional activity, has been confirmed several times. At present, P53 family proteins showing partial loss of activity appear to be promising potential candidates for the development of novel therapeutic strategies which could restore their transcriptional activity. In this context, it is important to employ tools to precisely monitor their activity; in relation to this, non-canonical DNA secondary structures in promoters including G-quadruplexes (G4s) were shown to influence the activity of transcription factors. Here, we used a defined yeast assay to evaluate the impact of differently modeled G4 forming sequences on a panel of partial function P53 family mutant proteins. Specifically, a 22-mer G4 prone sequence (derived from the KSHV virus) and five derivatives that progressively mutate characteristic guanine stretches were placed upstream of a minimal promoter, adjacent to a P53 response element in otherwise isogenic yeast luciferase reporter strains. The transactivation ability of cancer-associated P53 (TA-P53α: A161T, R213L, N235S, V272L, R282W, R283C, R337C, R337H, and G360V) or Ectodermal Dyplasia syndromes-related P63 mutant proteins (ΔN-P63α: G134D, G134V and inR155) were tested. Our results show that the presence of G4 forming sequences can increase the transactivation ability of partial function P53 family proteins. These observations are pointing to the importance of DNA structural characteristics for accurate classification of P53 family proteins functionality in the context of the wide variety of TP53 and TP63 germline and somatic mutations.
Assuntos
Quadruplex G , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/metabolismo , Ativação Transcricional , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , DNA/química , Proteínas Mutantes/genéticaRESUMO
TP63 germ-line mutations are responsible for a group of human ectodermal dysplasia syndromes, underlining the key role of P63 in the development of ectoderm-derived tissues. Here, we report the identification of two TP63 alleles, G134V (p.Gly173Val) and insR155 (p.Thr193_Tyr194insArg), associated to ADULT and EEC syndromes, respectively. These alleles, along with previously identified G134D (p.Gly173Asp) and R204W (p.Arg243Trp), were functionally characterized in yeast, studied in a mammalian cell line and modeled based on the crystal structure of the P63 DNA-binding domain. Although the p.Arg243Trp mutant showed both complete loss of transactivation function and ability to interfere over wild-type P63, the impact of p.Gly173Asp, p.Gly173Val, and p.Thr193_Tyr194insArg varied depending on the response element (RE) tested. Interestingly, p.Gly173Asp and p.Gly173Val mutants were characterized by a severe defect in transactivation along with interfering ability on two DN-P63α-specific REs derived from genes closely related to the clinical manifestations of the TP63-associated syndromes, namely PERP and COL18A1. The modeling of the mutations supported the distinct functional effect of each mutant. The present results highlight the importance of integrating different functional endpoints that take in account the features of P63 proteins' target sequences to examine the impact of TP63 mutations and the associated clinical variability.
Assuntos
Anodontia/genética , Mama/anormalidades , Fenda Labial/genética , Fissura Palatina/genética , Displasia Ectodérmica/genética , Obstrução dos Ductos Lacrimais/genética , Deformidades Congênitas dos Membros/genética , Mutação , Unhas Malformadas/genética , Transtornos da Pigmentação/genética , Elementos de Resposta , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Alelos , Substituição de Aminoácidos , Anodontia/metabolismo , Proteínas Reguladoras de Apoptose/genética , Mama/metabolismo , Linhagem Celular , Fenda Labial/metabolismo , Fissura Palatina/metabolismo , Displasia Ectodérmica/metabolismo , Regulação da Expressão Gênica , Estudos de Associação Genética , Mutação em Linhagem Germinativa , Células HCT116 , Humanos , Obstrução dos Ductos Lacrimais/metabolismo , Deformidades Congênitas dos Membros/metabolismo , Unhas Malformadas/metabolismo , Fenótipo , Transtornos da Pigmentação/metabolismo , Isoformas de Proteínas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Leveduras/genética , Leveduras/metabolismo , Proteína X Associada a bcl-2/genéticaRESUMO
Environmental and lifestyle exposures have a huge impact on cancer risk; nevertheless, the biological mechanisms underlying this association remain poorly understood. Extracellular vesicles (EVs) are membrane-enclosed particles actively released by all living cells, which play a key role in intercellular communication. EVs transport a variegate cargo of biomolecules, including non-coding RNA (ncRNA), which are well-known regulators of gene expression. Once delivered to recipient cells, EV-borne ncRNAs modulate a plethora of cancer-related biological processes, including cell proliferation, differentiation, and motility. In addition, the ncRNA content of EVs can be altered in response to outer stimuli. Such changes can occur either as an active attempt to adapt to the changing environment or as an uncontrolled consequence of cell homeostasis loss. In either case, such environmentally-driven alterations in EV ncRNA might affect the complex crosstalk between malignant cells and the tumor microenvironment, thus modulating the risk of cancer initiation and progression. In this review, we summarize the current knowledge about EV ncRNAs at the interface between environmental and lifestyle determinants and cancer. In particular, we focus on the effect of smoking, air and water pollution, diet, exercise, and electromagnetic radiation. In addition, we have conducted a bioinformatic analysis to investigate the biological functions of the genes targeted by environmentally-regulated EV microRNAs. Overall, we draw a comprehensive picture of the role of EV ncRNA at the interface between external factors and cancer, which could be of great interest to the development of novel strategies for cancer prevention, diagnosis, and therapy.
Assuntos
Vesículas Extracelulares , MicroRNAs , Neoplasias , Humanos , Fumar , Vesículas Extracelulares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/genética , Exposição Ambiental/efeitos adversos , Microambiente TumoralRESUMO
Aims: This retrospective study aimed to report clinical findings, treatment response and survival in cats diagnosed with precursor-targeted immune-mediated anaemia (PIMA) in a referral hospital in the UK. Methods: Feline cases diagnosed with presumed PIMA between January 2010 and February 2021 were retrospectively recruited. Signalment, clinical signs, diagnostic tests, treatment and outcome were recorded. Descriptive analytics were performed. Outcomes were documented according to survival to discharge, 30-day survival, total survival time, response to immunosuppressive treatment and frequency of relapses. Results: Thirty cats met the inclusion criteria. A higher prevalence of females (19/30) was identified (p = 0.001). Most cats (25/30) presented with haematocrit below 0.15 L/L. Concurrent cytopenias occurred in 18 of 30 cats. Bone marrow diagnosis was erythroid hyperplasia in 24 of 30 cases. Survival to discharge was documented in 26 of 30 cats, of which 23 survived more than 30 days since diagnosis. Initial treatment included blood products (26/30) and prednisolone (26/30) or prednisolone with ciclosporin (3/30); 18 of 30 cats responded to treatment, with a normal haematocrit at a mean of 28 days. The initial haematocrit and the presence of concurrent cytopenia were not statistically different between responders and non-responders. The median survival time was 140 days (range 1-3930 days). Conclusions and relevance: The treatment response rate of feline PIMA was high (60%), with a mortality rate of 23% over the 30 days following diagnosis. Relapses occur frequently (77%) but the response rate after treatment modification was high (76%) and therefore ongoing treatment may be justified at that point. Long survival times (up to 3930 days) can be achieved.