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BACKGROUND: The immune microenvironment impacts tumor growth, invasion, metastasis, and patient survival and may provide opportunities for therapeutic intervention in pancreatic ductal adenocarcinoma (PDAC). Although never studied as a potential modulator of the immune response in most cancers, Keratin 17 (K17), a biomarker of the most aggressive (basal) molecular subtype of PDAC, is intimately involved in the histogenesis of the immune response in psoriasis, basal cell carcinoma, and cervical squamous cell carcinoma. Thus, we hypothesized that K17 expression could also impact the immune cell response in PDAC, and that uncovering this relationship could provide insight to guide the development of immunotherapeutic opportunities to extend patient survival. METHODS: Multiplex immunohistochemistry (mIHC) and automated image analysis based on novel computational imaging technology were used to decipher the abundance and spatial distribution of T cells, macrophages, and tumor cells, relative to K17 expression in 235 PDACs. RESULTS: K17 expression had profound effects on the exclusion of intratumoral CD8+ T cells and was also associated with decreased numbers of peritumoral CD8+ T cells, CD16+ macrophages, and CD163+ macrophages (p < 0.0001). The differences in the intratumor and peritumoral CD8+ T cell abundance were not impacted by neoadjuvant therapy, tumor stage, grade, lymph node status, histologic subtype, nor KRAS, p53, SMAD4, or CDKN2A mutations. CONCLUSIONS: Thus, K17 expression correlates with major differences in the immune microenvironment that are independent of any tested clinicopathologic or tumor intrinsic variables, suggesting that targeting K17-mediated immune effects on the immune system could restore the innate immunologic response to PDAC and might provide novel opportunities to restore immunotherapeutic approaches for this most deadly form of cancer.
Assuntos
Queratina-17 , Neoplasias Pancreáticas , Humanos , Queratina-17/metabolismo , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Microambiente Tumoral/imunologia , Feminino , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Masculino , Linfócitos T CD8-Positivos/imunologia , Macrófagos/metabolismo , Macrófagos/imunologia , Pessoa de Meia-Idade , Idoso , Receptores de Superfície Celular , Antígenos de Diferenciação Mielomonocítica , Antígenos CDRESUMO
To date, most research on amyloid aggregation has focused on describing the structure of amyloids and the kinetics of their formation, while the conformational stability of fibrils remains insufficiently explored. The aim of this work was to investigate the effect of amino acid substitutions on the stability of apomyoglobin (ApoMb) amyloids. A study of the amyloid unfolding of ApoMb and its six mutant variants by urea has been carried out. Changes in the structural features of aggregates during unfolding were recorded by far-UV CD and native electrophoresis. It was shown that during the initial stage of denaturation, amyloids' secondary structure partially unfolds. Then, the fibrils undergo dissociation and form intermediate aggregates weighing approximately 1 MDa, which at the last stage of unfolding decompose into 18 kDa monomeric unfolded molecules. The results of unfolding transitions suggest that the stability of the studied amyloids relative to the intermediate aggregates and of the latter relative to unfolded monomers is higher for ApoMb variants with substitutions that increase the hydrophobicity of the residues. The results presented provide a new insight into the mechanism of stabilization of protein aggregates and can serve as a base for further investigations of the amyloids' stability.
Assuntos
Apoproteínas , Mioglobina , Substituição de Aminoácidos , Mioglobina/química , Estrutura Secundária de Proteína , Apoproteínas/química , Amiloide/genética , Dobramento de Proteína , Desnaturação ProteicaRESUMO
The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL14-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL14 which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL14 containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES.
Assuntos
Chaperoninas , Escherichia coli , Escherichia coli/metabolismo , Proteínas Luminescentes/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X , Chaperoninas/metabolismo , Dobramento de Proteína , Chaperonina 60/metabolismoRESUMO
Although oligomeric proteins are predominant in cells, their folding is poorly studied at present. This work is focused on the denaturant- and mutation-induced disassembly of the hexameric mutant Y55W of the Qß host factor (Hfq) from mesophilic Pseudomonas aeruginosa (Pae). Using intrinsic tryptophan fluorescence, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC), we show that the dissociation of Hfq Y55W occurs either under the effect of GuHCl or during the pre-denaturing transition, when the protein concentration is decreased, with both events proceeding through the accumulation of stable intermediate states. With an extremely low pH of 1.4, a low ionic strength, and decreasing protein concentration, the accumulated trimers and dimers turn into monomers. Also, we report on the structural features of monomeric Hfq resulting from a triple mutation (D9A/V43R/Y55W) within the inter-subunit surface of the protein. This globular and rigidly packed monomer displays a high thermostability and an oligomer-like content of the secondary structure, although its urea resistance is much lower.
Assuntos
Dobramento de Proteína , Pseudomonas aeruginosa , Dicroísmo Circular , Mutação , Desnaturação Proteica , Estrutura Secundária de Proteína , Termodinâmica , Triptofano/química , Ureia/farmacologiaRESUMO
BACKGROUND: Mutations in one allele of the TP53 gene in early stages are frequently followed by the loss of the remaining wild-type p53 (wtp53) allele (p53LOH) during tumor progression. Despite the strong notion of p53LOH as a critical step in tumor progression, its oncogenic outcomes that facilitate the selective pressure for p53LOH occurrence were not elucidated. METHODS: Using MMTV;ErbB2 mouse model of breast cancer carrying heterozygous R172H p53 mutation, we identified a novel gain-of-function (GOF) activity of mutant p53 (mutp53): the exacerbated loss of wtp53 allele in response to γ-irradiation. RESULTS: As consequences of p53LOH in mutp53 heterozygous cells, we observed profound stabilization of mutp53 protein, the loss of p21 expression, the abrogation of G2/M checkpoint, chromosomal instability, centrosome amplification, and transcriptional upregulation of mitotic kinase Nek2 (a member of Never in Mitosis (NIMA) Kinases family) involved in the regulation of centrosome function. To avoid the mitotic catastrophe in the absence of G2/M checkpoint, cells with centrosome amplification adapt Nek2-mediated centrosomes clustering as pro-survival mutp53 GOF mechanism enabling unrestricted proliferation and clonal expansion of cells with p53LOH. Thus, the clonal dominance of mutp53 cells with p53LOH may represent the mechanism of irradiation-induced p53LOH. We show that pharmacological and genetic ablation of Nek2 decreases centrosome clustering and viability of specifically mutp53 cells with p53LOH. CONCLUSION: In a heterogeneous tumor population, Nek2 inhibition may alter the selective pressure for p53LOH by contraction of the mutp53 population with p53LOH, thus, preventing the outgrowth of genetically unstable, more aggressive cells.
Assuntos
Neoplasias da Mama/genética , Perda de Heterozigosidade/genética , Quinases Relacionadas a NIMA/genética , Proteína Supressora de Tumor p53/genética , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Centrossomo/metabolismo , Instabilidade Cromossômica , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Mutação com Ganho de Função , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Perda de Heterozigosidade/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Quinases Relacionadas a NIMA/antagonistas & inibidores , Receptor ErbB-2/genética , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
The discovery of a subunit exchange in some oligomeric proteins, implying short-term dissociation of their oligomeric structure, requires new insights into the role of the quaternary structure in oligomeric protein stability and function. Here we demonstrate the effect of pH, protein concentration, and urea on the efficiency of GroES heptamer (GroES7) subunit exchange. A mixture of equimolar amounts of wild-type (WT) GroES7 and its Ala97Cys mutant modified with iodoacetic acid (97-carboxymethyl cysteine or CMC-GroES7) was incubated in various conditions and subjected to isoelectric focusing (IEF) in polyacrylamide gel. For each sample, there are eight Coomassie-stained electrophoretic bands showing different charges that result from a different number of included mutant subunits, each carrying an additional negative charge. The intensities of these bands serve to analyze the protein subunit exchange. The protein stability is evaluated using the transverse urea gradient gel electrophoresis (TUGGE). At pH 8.0, the intensities of the initial bands corresponding to WT-GroES7 and CMC-GroES7 are decreased with a half-time of (23 ± 2) min. The exchange decreases with decreasing pH and seems to be strongly hindered at pH 5.2 due to the protonation of groups with pK â¼ 6.3, which stabilizes the protein quaternary structure. The destabilization of the protein quaternary structure caused by increased pH, decreased protein concentration, or urea accelerates the GroES subunit exchange. This study allows visualizing the subunit exchange in oligomeric proteins and confirms its direct connection with the stability of the protein quaternary structure.
Assuntos
Ureia , Concentração de Íons de Hidrogênio , Ureia/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Estabilidade Proteica , Focalização IsoelétricaRESUMO
OBJECTIVES: To determine the role of keratin 17 (K17) as a predictive biomarker for response to chemotherapy by defining thresholds of K17 expression based on immunohistochemical tests that could be used to optimize therapeutic intervention for patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: We profiled K17 expression, a hallmark of the basal molecular subtype of PDAC, by immunohistochemistry in 2 cohorts of formalin-fixed, paraffin-embedded PDACs (n = 305). We determined a K17 threshold of expression to optimize prognostic stratification according to the lowest Akaike information criterion and explored the potential relationship between K17 and chemoresistance by multivariate predictive analyses. RESULTS: Patients with advanced-stage, low K17 PDACs treated using 5-fluorouracil (5-FU)-based chemotherapeutic regimens had 3-fold longer survival than corresponding cases treated with gemcitabine-based chemotherapy. By contrast, PDACs with high K17 did not respond to either regimen. The predictive value of K17 was independent of tumor mutation status and other clinicopathologic variables. CONCLUSIONS: The detection of K17 in 10% or greater of PDAC cells identified patients with shortest survival. Among patients with low K17 PDACs, 5-FU-based treatment was more likely than gemcitabine-based therapies to extend survival.
RESUMO
Background: The immune microenvironment impacts tumor growth, invasion, metastasis, and patient survival and may provide opportunities for therapeutic intervention in pancreatic ductal adenocarcinoma (PDAC). Although never studied as a potential modulator of the immune response in most cancers, Keratin 17 (K17), a biomarker of the most aggressive (basal) molecular subtype of PDAC, is intimately involved in the histogenesis of the immune response in psoriasis, basal cell carcinoma, and cervical squamous cell carcinoma. Thus, we hypothesized that K17 expression could also impact the immune cell response in PDAC, and that uncovering this relationship could provide insight to guide the development of immunotherapeutic opportunities to extend patient survival. Methods: Multiplex immunohistochemistry (mIHC) and automated image analysis based on novel computational imaging technology were used to decipher the abundance and spatial distribution of T cells, macrophages, and tumor cells, relative to K17 expression in 235 PDACs. Results: K17 expression had profound effects on the exclusion of intratumoral CD8 + T cells and was also associated with decreased numbers of peritumoral CD8 + T cells, CD16 + macrophages, and CD163 + macrophages (p < 0.0001). The differences in the intratumor and peritumoral CD8 + T cell abundance were not impacted by neoadjuvant therapy, tumor stage, grade, lymph node status, histologic subtype, nor KRAS, p53, SMAD4, or CDKN2A mutations. Conclusions: Thus, K17 expression correlates with major differences in the immune microenvironment that are independent of any tested clinicopathologic or tumor intrinsic variables, suggesting that targeting K17-mediated immune effects on the immune system could restore the innate immunologic response to PDAC and might provide novel opportunities to restore immunotherapeutic approaches for this most deadly form of cancer.
RESUMO
Cytoprotective heme oxygenases derivatize heme to generate carbon monoxide, ferrous iron, and isomeric biliverdins, followed by rapid NAD(P)H-dependent biliverdin reduction to the antioxidant bilirubin. Recent studies have implicated biliverdin IXß reductase (BLVRB) in a redox-regulated mechanism of hematopoietic lineage fate restricted to megakaryocyte and erythroid development, a function distinct and non-overlapping from the BLVRA (biliverdin IXα reductase) homologue. In this review, we focus on recent progress in BLVRB biochemistry and genetics, highlighting human, murine, and cell-based studies that position BLVRB-regulated redox function (or ROS accumulation) as a developmentally tuned trigger that governs megakaryocyte/erythroid lineage fate arising from hematopoietic stem cells. BLVRB crystallographic and thermodynamic studies have elucidated critical determinants of substrate utilization, redox coupling and cytoprotection, and have established that inhibitors and substrates bind within the single-Rossmann fold. These advances provide unique opportunities for the development of BLVRB-selective redox inhibitors as novel cellular targets that retain potential for therapeutic applicability in hematopoietic (and other) disorders.
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Contrary to high doses irradiation (HDR), the biological consequences of dose irradiation (LDR) in breast cancer remain unclear due to the complexity of human epidemiological studies. LDR induces DNA damage that activates p53-mediated tumor-suppressing pathways promoting DNA repair, cell death, and growth arrest. Monoallelic p53 mutations are one of the earliest and the most frequent genetic events in many subtypes of cancer including ErbB2 breast cancer. Using MMTV/ErbB2 mutant p53 (R172H) heterozygous mouse model we found differential p53 genotype-specific effect of LDR vs. HDR on mammary tumorigenesis. Following LDR, mutant p53 heterozygous tumor cells exhibit aberrant ATM/DNA-PK signaling with defects in sensing of double-strand DNA brakes and deficient DNA repair. In contrast, HDR-induced genotoxic stress is sufficient to reach the threshold of DNA damage that is necessary for wtp53 induced DNA repair and cell cycle arrest. As a result, mutant p53 endows dominant-negative effect promoting mammary tumorigenesis after low-impact DNA damage leading to the selection of a genetically unstable proliferative population, with negligible mutagenic effect on tumors carrying wtp53 allele.
Assuntos
Raios gama/uso terapêutico , Mutação/efeitos da radiação , Proteína Supressora de Tumor p53/genética , Animais , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/efeitos da radiação , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Proteína Quinase Ativada por DNA/genética , Feminino , Camundongos , Mutação/genética , Receptor ErbB-2/genéticaRESUMO
The production of recombinant proteins in Escherichia coli cells is often hampered by aggregation of newly synthesized proteins and formation of inclusion bodies. Here we propose the use of transverse urea gradient electrophoresis (TUGE) in testing the capability of folding of a recombinant protein from inclusion bodies dissolved in urea. A plasmid encoding the amino acid sequence 55-224 of TcpA pilin (C-terminal globular domain: TcpA-C) from Vibrio cholerae El Tor enlarged by a His-tag on its N-terminus was expressed in E. coli cells. The major fraction (about 90%) of the target polypeptide was detected in cell debris. The polypeptide was isolated from the soluble fraction and recovered from inclusion bodies after their urea treatment. Some structural properties of the polypeptide from each sample proved identical. The refolding protocol was developed on the basis of TUGE data and successfully used for the protein large-scale recovery from inclusion bodies. Spectral, hydrodynamic, and thermodynamic characteristics of the recombinant TcpA recovered from inclusion bodies indicate the presence of a globular conformation with a pronounced secondary structure and a rigid tertiary structure, which is promising for the design of immunodiagnostics preparations aimed to assess the pilin level in different strains of V. cholerae and to develop cholera vaccines.
RESUMO
The main events in chaperone-assisted protein folding are the binding and ligand-induced release of substrate proteins. Here, we studied the location of denatured proteins previously bound to the GroEL chaperonin resulting from the action of the GroES co-chaperonin in the presence of Mg-ATP. Fluorescein-labeled denatured proteins (α-lactalbumin, lysozyme, serum albumin, and pepsin in the presence of thiol reagents at neutral pH, as well as an early refolding intermediate of malate dehydrogenase) were used to reveal the effect of GroES on their interaction with GroEL. Native electrophoresis has demonstrated that these proteins tend to be released from the GroEL-GroES complex. With the use of biotin- and fluorescein-labeled denatured proteins and streptavidin fused with luciferase aequorin (the so-called streptavidin trap), the presence of denatured proteins in bulk solution after GroES and Mg-ATP addition has been confirmed. The time of GroES-induced dissociation of a denatured protein from the GroEL surface was estimated using the stopped-flow technique and found to be much shorter than the proposed time of the GroEL ATPase cycle.
Assuntos
Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Dobramento de Proteína , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Ligação Proteica , Desnaturação Proteica , Mapas de Interação de ProteínasRESUMO
Mutations in one allele of the TP53 gene in cancer early stages are frequently followed by the loss of the remaining wild-type allele (LOH) during tumor progression. However, the clinical impact of TP53 mutations and p53LOH, especially in the context of genotoxic modalities, remains unclear. Using MMTV;ErbB2 model carrying a heterozygous R172H p53 mutation, we report a previously unidentified oncogenic activity of mutant p53 (mutp53): the exacerbation of p53LOH after irradiation. We show that wild-type p53 allele is partially transcriptionally competent and enables the maintenance of the genomic integrity under normal conditions in mutp53 heterozygous cells. In heterozygous cells γ-irradiation promotes mutp53 stabilization, which suppresses DNA repair and the cell cycle checkpoint allowing cell cycle progression in the presence of inefficiently repaired DNA, consequently increases genomic instability leading to p53LOH. Hence, in mutp53 heterozygous cells, irradiation facilitates the selective pressure for p53LOH that enhances cancer cell fitness and provides the genetic plasticity for acquiring metastatic properties.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Perda de Heterozigosidade/efeitos da radiação , Mutação , Proteína Supressora de Tumor p53/genética , Animais , Biomarcadores Tumorais , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/efeitos da radiação , Dano ao DNA , Modelos Animais de Doenças , Feminino , Raios gama , Instabilidade Genômica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Camundongos , Camundongos Transgênicos , Estadiamento de Neoplasias , Prognóstico , Resultado do TratamentoRESUMO
Despite success of ERBB2-targeted therapies such as lapatinib, resistance remains a major clinical concern. Multiple compensatory receptor tyrosine kinase (RTK) pathways are known to contribute to lapatinib resistance. The heterogeneity of these adaptive responses is a significant hurdle for finding most effective combinatorial treatments. The goal of this study was to identify a unifying molecular mechanism whose targeting could help prevent and/or overcome lapatinib resistance. Using the MMTV-ERBB2;mutant p53 (R175H) in vivo mouse model of ERBB2-positive breast cancer, together with mouse and human cell lines, we compared lapatinib-resistant vs. lapatinib-sensitive tumor cells biochemically and by kinome arrays and evaluated their viability in response to a variety of compounds affecting heat shock response. We found that multiple adaptive RTKs are activated in lapatinib-resistant cells in vivo, some of which have been previously described (Axl, MET) and some were novel (PDGFRα, PDGFRß, VEGFR1, MUSK, NFGR). Strikingly, all lapatinib-resistant cells show chronically activated HSF1 and its transcriptional targets, heat shock proteins (HSPs), and, as a result, superior tolerance to proteotoxic stress. Importantly, lapatinib-resistant tumors and cells retained sensitivity to Hsp90 and HSF1 inhibitors, both in vitro and in vivo, thus providing a unifying and actionable therapeutic node. Indeed, HSF1 inhibition simultaneously downregulated ERBB2, adaptive RTKs and mutant p53, and its combination with lapatinib prevented development of lapatinib resistance in vitro. Thus, the kinome adaptation in lapatinib-resistant ERBB2-positive breast cancer cells is governed, at least in part, by HSF1-mediated heat shock pathway, providing a novel potential intervention strategy to combat resistance.
Assuntos
Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fatores de Transcrição de Choque Térmico/metabolismo , Lapatinib/farmacologia , Receptor ErbB-2/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Proteólise/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mutations in the p53 tumor suppressor gene are the most prevalent genetic events in human Her2-positive breast cancer and are associated with poor prognosis and survival. Human clinical data and our in vitro and in vivo studies strongly suggest potent oncogenic cooperation between mutant p53 and Her2 (ErbB2). Yet, the translational significance of mutant p53 in Her2 positive breast cancer, especially with respect to Her2-targeted therapies, has not been evaluated. Our previous work identified novel oncogenic activity of mutant p53 whereby mutp53 amplifies ErbB2 signaling via the mutp53-HSF1-ErbB2 feed-forward loop. Here we report that pharmacological interception of this circuit by ErbB2 inhibitor lapatinib downregulates mutant p53 in vitro and in vivo. We found that ErbB2 inhibition by lapatinib inhibits transcription factor HSF1, and its target Hsp90, followed by mutant p53 degradation in MDM2 dependent manner. Thus, our data suggest that mutant p53 sensitizes cancer cells to lapatinib via two complementary mechanisms: mutant p53 mediated amplification of ErbB2 signaling, and simultaneous annihilation of both potent oncogenic drivers, ErbB2 and mutant p53. Hence, our study could provide valuable information for the optimization of therapeutic protocols to achieve superior clinical effects in the treatment of Her2 positive breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Quinazolinas/administração & dosagem , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lapatinib , Camundongos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteólise , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Missense mutations in TP53 comprise >75% of all p53 alterations in cancer, resulting in highly stabilized mutant p53 proteins that not only lose their tumor-suppressor activity, but often acquire oncogenic gain-of-functions (GOFs). GOF manifests itself in accelerated tumor onset, increased metastasis, increased drug resistance and shortened survival in patients and mice. A known prerequisite for GOF is mutant p53 protein stabilization, which itself is linked to aberrant protein conformation. However, additional determinants for mutant p53 stabilization likely exist. Here we show that in initially heterozygous mouse tumors carrying the hotspot GOF allele R248Q (p53Q/+), another necessary prerequisite for mutant p53 stabilization and GOF in vivo is loss of the remaining wild-type p53 allele, termed loss-of-heterozygosity (LOH). Thus, in mouse tumors with high frequency of p53 LOH (osteosarcomas and fibrosarcomas), we find that mutant p53 protein is stabilized (16/17 cases, 94%) and tumor onset is significantly accelerated compared with p53+/- tumors (GOF). In contrast, in mouse tumors with low frequency of p53 LOH (MMTV-Neu breast carcinomas), mutant p53 protein is not stabilized (16/20 cases, 80%) and GOF is not observed. Of note, human genomic databases (TCGA, METABRIC etc.) show a high degree of p53 LOH in all examined tumor types that carry missense p53 mutations, including sarcomas and breast carcinomas (with and without HER2 amplification). These data - while cautioning that not all genetic mouse models faithfully represent the human situation - demonstrate for the first time that p53 LOH is a critical prerequisite for missense mutant p53 stabilization and GOF in vivo.
Assuntos
Neoplasias da Mama/genética , Perda de Heterozigosidade/genética , Proteína Supressora de Tumor p53/genética , Alelos , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Mutação de Sentido Incorreto/genéticaRESUMO
A quantitative assessment of rate-limiting steps in metastasis has always been challenging because of the difficulty of detecting small tumor cell populations. We have developed a highly sensitive assay for monitoring the metastatic dissemination of human tumor cells in the chick embryo and used this assay to investigate the relative efficacy of sequential stages in the metastatic cascade for two malignant human tumor cells lines, HEp3 and HT1080. This assay is based on the real-time PCR amplification of human alu sequences and exhibits a high sensitivity (25 cells/lung) with a large linear range (50-100,000 cell/lung). The assay is optimized for a high number of replicate in vivo assays (50-100 animals/assay) and can be applied in both experimental and spontaneous metastasis models. Using quantitative alu PCR, we determined that HEp3 spontaneously metastasizes very efficiently and rapidly, generating secondary growth in the lung exceeding 1-2 x 10(4) cells/lung in 7 days. In contrast, spontaneous HT1080 metastasis is 50-100-fold less efficient, resulting in only 200-400 cells/lung in 7 days. By taking advantage of the sensitivity and specificity of the real-time alu PCR assay we were also able to quantitatively assess multiple steps in metastasis including intravasation, arrest of tumor cells in secondary organs of the embryo, and the initial growth and expansion of the arrested tumor cells. A comparative analysis of HEp3 and HT1080 metastasis demonstrates that the relatively low-to-moderate metastatic rate of HT1080 is caused by two distinct deficiencies, an 8-10-fold lower rate of intravasation and a delayed onset of HT1080 growth expansion in the secondary organ. Thus, a very facile metastasis model system coupled with the sensitive, real-time PCR-based assay allows for the identification and quantification of rate-limiting steps in the metastatic cascade for select human tumor cell lines.
Assuntos
Metástase Neoplásica/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Animais , Embrião de Galinha , Fibrossarcoma/patologia , Fibrossarcoma/secundário , Células HeLa , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundárioRESUMO
SUMMARY: The aim of the article was to study changes in periodontal tissues in rats with spontaneous periodontitis (SP) and to evaluate the effect of hyaluronic acid (HA) on the state of the periodontium. Wistar rats with signs of SP were divided into 6 groups: 1) intact group; 2) intact animals with HA "HD-1,0 MDa"; 3) SP group; 4) SP with HA "S-2,4 MDa"; 5) SP with HA "ST-2,4 MDa"; 6) SP with HA "HD-1,0 MDa". The study of the periodontium rats with SP noted the main structural changes (collagen reduction, resorption of alveolar bone, dilatation and stasis of the vessels of the periodontium, gingival papilla and tooth pulp), which were assessed as moderate. Morphological evidence of inflammation was infiltration of neutrophils into the connective tissue of the gums, without the formation of abscesses. Local administration of HA did not cause additional structural damage in periodontal tissues of rats with SP, but also did not affect changes in the microvascular system of periodontium and tooth pulp, periodontal ligaments, only a tendency to inhibit alveolar bone resorption in rats was noted. One can consider the tendency to improve the condition of periodontal tissues in the group of rats injected with high molecular HA and HA with mannitol (2.4 MDa).
RESUMEN: El objetivo del artículo fue estudiar los cambios en los tejidos periodontales en ratas con periodontitis espontánea (PE) y evaluar el efecto del ácido hialurónico (HA) sobre el estado del periodonto. Las ratas Wistar con signos de PE se dividieron en 6 grupos: 1) grupo intacto; 2) animales intactos con HA "HD-1,0 MDa"; 3) grupo PE; 4) PE con HA "S-2,4 MDa"; 5) PE con HA "ST-2,4 MDa"; 6) PE con HA "HD-1,0 MDa". En las ratas con PS se observaron los principales cambios estructurales (reducción de colágeno, reabsorción del hueso alveolar, dilatación y estasis de los vasos del periodonto, papila gingival y pulpa dentaria), que fueron evaluados como moderados. La evidencia morfológica de inflamación fue la infiltración de neutrófilos en el tejido conectivo de las encías, sin la formación de abscesos. La administración local de HA no causó daño estructural adicional en los tejidos periodontales de las ratas con PE, pero tampoco se produjo cambios en el sistema microvascular del periodonto y en la pulpa dental y ligamentos periodontales.Se observó una tendencia a inhibir la resorción del hueso alveolar. Se puede considerar la tendencia a mejorar el estado de los tejidos periodontales en el grupo de ratas inyectadas con HA de alto peso molecular y HA con manitol (2,4 MDa).
Assuntos
Animais , Ratos , Periodontite , Periodonto/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Ratos Wistar , InflamaçãoRESUMO
The main tumor suppressor function of p53 as a "guardian of the genome" is to respond to cellular stress by transcriptional activation of apoptosis, growth arrest, or senescence in damaged cells. Not surprisingly, mutations in the p53 gene are the most frequent genetic alteration in human cancers. Importantly, mutant p53 (mutp53) proteins not only lose their wild-type tumor suppressor activity but also can actively promote tumor development. Two main mechanisms accounting for mutp53 proto-oncogenic activity are inhibition of the wild-type p53 in a dominant-negative fashion and gain of additional oncogenic activities known as gain-of-function (GOF). Here, we discuss a novel mechanism of mutp53 GOF, which relies on its oncogenic cooperation with the heat shock machinery. This coordinated adaptive mechanism renders cancer cells more resistant to proteotoxic stress and provides both, a strong survival advantage to cancer cells and a promising means for therapeutic intervention.