RESUMO
BACKGROUND: Therapeutic drug monitoring (TDM) is routinely used for optimization of vancomycin therapy, because of exposure-related efficacy and toxicity, in addition to significant variability in pharmacokinetics, which leads to unpredictable drug exposure. OBJECTIVE: The aim of this study was to evaluate target attainment and TDM of vancomycin in neonates. METHODS: The authors conducted a retrospective study and collected data from medical records of all neonates who received vancomycin therapy in the neonatal intensive care unit between January 2019 and December 2019. The primary outcome was the proportion of vancomycin courses that reached target trough concentrations of 10-20 mg/L based on appropriate TDM samples collection. Secondary outcomes included proportion of courses with appropriate dose and dose frequency, and proportion of patients who achieved target concentrations after the first dose adjustment. RESULTS: In total, 69 patients were included, with 129 vancomycin courses. The median initial vancomycin trough concentration was 12 (range: 4-36) mg/L. The target trough concentration was achieved in 75% of courses after the initial dose with appropriate TDM, and 84% of courses after TDM-guided dose adjustments. Patients were dosed appropriately in 121/129 courses and TDM was performed correctly according to protocol in 51/93 courses. A dose adjustment was performed in 18/29 courses, to increase target attainment. CONCLUSIONS: This study showed that there is a need for an increase in dose to improve target attainment. There is also a need to explore more effective TDM strategies to increase the proportion of neonatal patients attaining vancomycin target trough concentrations.
Assuntos
Monitoramento de Medicamentos , Vancomicina , Antibacterianos/farmacocinética , Monitoramento de Medicamentos/métodos , Humanos , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Estudos Retrospectivos , Vancomicina/farmacocinéticaRESUMO
BACKGROUND: Tetrapyrrole substrates and products of heme oxygenase are potent inhibitors of hepatitis C virus (HCV) replication. It is not clear whether this occurs through primary induction of type I interferon (IFN), inhibition of viral NS3/4A protease, or a combination of these mechanisms. We studied the antiviral actions of tetrapyrroles and their potential influence on type I IFN induction. METHODS: The effects of tetrapyrrole on NS3/4A protease activity and type I IFN induction were assessed in HCV-permissive cells, replicons, or human embryonic kidney (HEK) 293 cells transfected with NS3/4A protease. Activation of innate immune signaling was determined after transfection of double-strand surrogate nucleic acid antigens or infection with defined sequence HCV cell culture (HCVcc) RNA. RESULTS: Tetrapyrroles failed to directly induce IFN expression at concentrations that inhibited HCV replication and NS3/4A protease activity. However, they potently restored IFN induction after attenuation with NS3/4A protease, a process accompanied by preservation of the adapter protein, mitochondrial antiviral signaling protein, nuclear localization of IFN regulatory factor 3, and augmentation of IFN-stimulated gene products. CONCLUSIONS: Tetrapyrroles do not directly induce IFN, but they dramatically restore type I IFN signaling pathway after attenuation with NS3/4A protease. They show immunomodulatory as well as antiprotease activity and may be useful for treatment of HCV infection.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Heme/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Interferon Tipo I/biossíntese , Tetrapirróis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/farmacologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Inibidores de Proteases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
In the later-line setting or for patients with PD-L1-negative tumors, immunotherapy-based regimens remain ineffective against advanced triple-negative breast cancer (TNBC). In this multicentered phase II trial (NCT04303741), 46 patients with pretreated advanced TNBC were enrolled to receive camrelizumab 200 mg (day 1), and apatinib 250 mg daily, plus eribulin 1.4 mg/m2 (day 1 and 8) on a 21-day cycle until progression, or unacceptable toxicity. Primary endpoint was objective response rate (ORR) according to RECIST 1.1. Secondary endpoints included toxicities, disease control rate (DCR), clinical benefit rate, progression-free survival (PFS), and 1-year overall survival. With a median of 3 lines of prior chemotherapy in the advanced setting, 17.4% had received PD-1/PD-L1 blockade plus chemotherapy for advanced disease. The ORR was 37.0% (17/46, 95% CI 23.2-52.5). The DCR was 87.0% (40/46, 95% CI 73.7-95.1). Median PFS was 8.1 (95% CI 4.6-10.3) months. Tertiary lymphoid structure was associated with higher ORR. Patients with lower tumor PML or PLOD3 expression had favorable ORR and PFS. PD-L1 status was not associated with ORR/PFS. Grade 3/4 treatment-related adverse events occurred in 19 (41.3%) of 46 patients. Camrelizumab plus apatinib and eribulin shows promising efficacy with a measurable safety profile in patients with heavily pretreated advanced TNBC.
Assuntos
Neoplasias de Mama Triplo Negativas , Anticorpos Monoclonais Humanizados , Antígeno B7-H1 , Furanos , Humanos , Cetonas , Piridinas , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
UNLABELLED: Induction of heme oxygenase-1 (HO-1) inhibits hepatitis C virus (HCV) replication. Of the products of the reaction catalyzed by HO-1, iron has been shown to inhibit HCV ribonucleic acid (RNA) polymerase, but little is known about the antiviral activity of biliverdin (BV). Herein, we report that BV inhibits viral replication and viral protein expression in a dose-dependent manner in replicons and cells harboring the infectious J6/JFH construct. Using the SensoLyte 620 HCV Protease Assay with a wide wavelength excitation/emission (591 nm/622 nm) fluorescence energy transfer peptide, we found that both recombinant and endogenous nonstructural 3/4A (NS3/4A) protease from replicon microsomes are potently inhibited by BV. Of the tetrapyrroles tested, BV was the strongest inhibitor of NS3/4A activity, with a median inhibitory concentration (IC(50)) of 9 µM, similar to that of the commercial inhibitor, AnaSpec (Fremont, CA) #25346 (IC(50) 5 µM). Lineweaver-Burk plots indicated mixed competitive and noncompetitive inhibition of the protease by BV. In contrast, the effects of bilirubin (BR) on HCV replication and NS3/4A were much less potent. Because BV is rapidly converted to BR by biliverdin reductase (BVR) intracellularly, the effect of BVR knockdown on BV antiviral activity was assessed. After greater than 80% silencing of BVR, inhibition of viral replication by BV was enhanced. BV also increased the antiviral activity of α-interferon in replicons. CONCLUSION: BV is a potent inhibitor of HCV NS3/4A protease, which likely contributes to the antiviral activity of HO-1. These findings suggest that BV or its derivatives may be useful in future drug therapies targeting the NS3/4A protease.
Assuntos
Antivirais/farmacologia , Biliverdina/farmacologia , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Linhagem Celular Tumoral , Heme Oxigenase-1/farmacologia , Hepacivirus/efeitos dos fármacos , Humanos , Cinética , Inibidores de Serina Proteinase , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacosRESUMO
Zinc protoporphyrin (ZnPP), a naturally occurring metalloprotoporphyrin (MPP), is currently under development as a chemotherapeutic agent although its mechanism is unclear. When tested against other MPPs, ZnPP was the most effective DNA synthesis and cellular proliferation inhibitor while promoting apoptosis in telomerase positive but not telomerase negative cells. Concurrently, ZnPP down-regulated telomerase expression and was the best overall inhibitor of telomerase activity in intact cells and cellular extracts with IC50 and EC50 values of ca 2.5 and 6 µM, respectively. The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non-denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (>600 kD) that contained telomerase and dysskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co-localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S-phase cells. ZnPP also co-localized with TERT in the perinuclear regions of log phase cells but did not co-localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Overall, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase activity. In conclusion, ZnPP actively interferes with telomerase activity in neoplastic cells, thus promoting pro-apoptotic and anti-proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for neoplastic disease.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Protoporfirinas/farmacologia , Telomerase/metabolismo , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Células HEK293 , Humanos , Concentração Inibidora 50 , Microscopia Confocal , Protoporfirinas/administração & dosagem , Telomerase/antagonistas & inibidores , Telômero/metabolismoRESUMO
Hepatitis C is an oncogenic virus although the mechanisms responsible for this behavior are not clear. We studied the effects of hepatitis C virus (HCV) core protein expression on Telomerase, an enzyme closely associated with cellular immortalization and neoplasia. The aim of this study was to investigate the effects of HCV core protein on the regulation of Telomerase activity in human hepatoma cells. Regulation and expression of human Telomerase reverse transcriptase (TERT) was compared in Huh7 cells stably transfected with HCV core protein or cells expressing vector alone. Telomerase activity was measured using Quantitative Telomerase Detection (QTD) and telomere length was measured by fluorescence in situ hybridization (FISH). Transient transfection and luciferase assay were used to evaluate TERT promoter activity. Telomerase activity was increased twofold in Huh7 cells expressing HCV core protein compared to controls (P < 0.01). This was accompanied by a 1.4-fold increase of TERT mRNA and 1.9-fold increase in TERT protein (P < 0.01 in either case). Cellular fractionation and immunocytochemical studies showed increased localization of TERT in the nucleus of core-expressing cells as compared to controls. FISH assay confirmed that telomeres of HCV core-expressing Huh7 cells were relatively longer than those of control cells (0.22 + 0.05 vs. 0.12 + 0.03, P < 0.01). TERT promoter activity was enhanced about 30% in HCV core-expressing Huh7 cells compared to control cells (P < 0.02). HCV core protein is associated with increased Telomerase activity in hepatoma cells. These findings suggest that enhancement of Telomerase activity by HCV core protein may contribute to the oncogenicity of HCV.
Assuntos
Hepacivirus/patogenicidade , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Telomerase/biossíntese , Proteínas do Core Viral/metabolismo , Fusão Gênica Artificial , Linhagem Celular Tumoral , Núcleo Celular/química , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Hibridização in Situ Fluorescente/métodos , Luciferases/biossíntese , Luciferases/genética , Telômero/genética , Regulação para CimaRESUMO
Clear cell renal cell carcinoma (ccRCC) is a very common cancer in urology. Many evidences suggest that complex changed pathways take a nonnegligible part in the occurrence and development of ccRCC. Nevertheless, the underlying mechanism is not clear. In this study, the expression data between ccRCC and normal tissue samples in TCGA database were compared to distinguish differentially expressed genes (DEGs: mRNAs, miRNAs, and lncRNAs). Afterwards, we used GO enrichment and KEGG pathway enrichment analyses to explore the functions of these DEGs. We also found the correlation between three RNAs and created a competing endogenous RNA (ceRNA) network. Moreover, we used univariate Cox regression analysis to select DEGs that are connected with overall survival (OS) of ccRCC patients. We found 1652 mRNAs, 1534 lncRNAs, and 173 miRNAs that were distinguished in ccRCC compared with normal tissues. According to GO analysis, the maladjusted mRNAs are mainly concentrated in immune cell activation and kidney development, while according to KEGG, they are mainly concentrated in pathways related to cancer. A total of 5 mRNAs, 1 miRNA, and 4 lncRNAs were connected with patients' OS. In this article, a network of lncRNA-miRNA-mRNA was established; it is expected to be able to indicate possible molecular mechanisms for initial of ccRCC and provide a new viewpoint for diagnosis of ccRCC.
Assuntos
Carcinoma de Células Renais , Bases de Dados de Ácidos Nucleicos , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais , RNA Viral , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , Masculino , Valor Preditivo dos Testes , RNA Viral/biossíntese , RNA Viral/genética , Taxa de SobrevidaRESUMO
BACKGROUND: Antiviral actions of tetrapyrroles have been described in a number of systems. Our goal was to evaluate antagonism of the HCV NS3-4A protease by a variety of common porphyrins and characterize structure-activity relationships that may be useful for future drug design of HCV and related Flaviviruses. METHODS: Using fluorometric assays, common metalloprotoporphyrins (MPP) all inhibited NS3-4A protease with IC50 values in low micromolar ranges [CoPP (1.4 µM) < ZnPP = MnPP = SnPP < CuPP < FePP (6.5 µM) = protoporphyrin]. RESULTS: Lineweaver-Burk plots confirmed that MPP: NS3 inhibition was basically competitive. All tested MPPs inhibited HCV genotype 1A, 1B, 2A and 3A recombinant proteases with the same fidelity suggesting wide antagonistic capabilities. However, when the MPPs were tested in cellular incubations with HCV replicons only Zn, Fe and free-base protoporphyrin showed comparable EC50 and IC50 values suggesting that there may be critical differences in MPP uptake and intracellular availability. Meso, deutero, and isohematoporphyrin derivatives, with or without metal substitution, all showed less anti-protease and antiviral activities as compared to protoporphyrins, suggesting that the planar, vinyl side chains are important for protease active site binding. MPPs were also active against three common protease mutants (T54A, A156T, and V36M) with equivalent or better IC50 values as compared to wild type enzyme. CONCLUSION: These findings document the versatility of MPPs as antiviral agents with an expanded sensitivity for HCV genotypes and resistance to some common viral mutations. The results also suggest that further study of MPP structure and function will be useful for the development of new antiviral agents.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , Células Cultivadas , Relação Dose-Resposta a Droga , Hepacivirus/genética , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Inibidores de Proteases/química , Serina Proteases/genética , Serina Proteases/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
UNLABELLED: Oxidative injury to hepatocytes occurs as a result of hepatitis C virus (HCV) infection and replication. Modulation of host cell antioxidant enzymes such as heme oxygenase-1 (HO-1) may be useful therapeutically to minimize cellular injury, reduce viral replication, and attenuate liver disease. In this report, we evaluated the effects of HO-1 overexpression on HCV replication and hepatocellular injury. Full-length (FL) (Con1) or nonstructural (NS) replicons (I 389 NS3-3') were transfected with complete human HO-1 sequences or empty vector for control. Cell lines overexpressing HO-1 (twofold to sixfold above basal values) or empty vector were isolated, and their HCV RNA synthesis, pro-oxidant levels, and resistance to oxidative injury were assessed. HO-1 overexpression decreased HCV RNA replication in both FL and NS replicons without affecting cellular growth or DNA synthesis. The attenuation of HCV replication was significantly reversed in both replicon systems with HO-1 small interfering RNA (siRNA) knockdown. Both FL and NS replicons that overexpress HO-1 showed reduced prooxidant levels at baseline and increased resistance to oxidant-induced cytotoxicity. HO-1 induction with hemin also markedly decreased HCV replication in both parental FL and NS replicon cell lines. Conversely, knockdown of HO-1 messenger RNA (mRNA) by siRNA in parental FL or NS replicons did not significantly affect HCV replication, suggesting that less than basal levels of HO-1 had minimal effect on HCV replication. CONCLUSION: Overexpression or induction of HO-1 results in decreased HCV replication as well as protection from oxidative damage. These findings suggest a potential role for HO-1 in antiviral therapy and therapeutic protection against hepatocellular injury in HCV infection.
Assuntos
Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Hepacivirus/fisiologia , Hepatócitos/enzimologia , Oxidantes/toxicidade , Carcinoma Hepatocelular , Divisão Celular , Linhagem Celular Tumoral , Primers do DNA , Heme Oxigenase-1/deficiência , Hepacivirus/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Neoplasias Hepáticas , RNA Interferente Pequeno/genética , Replicon/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Replicação Viral/efeitos dos fármacos , terc-Butil Hidroperóxido/farmacologiaRESUMO
Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along the MAPK pathway are central for transformation. BRAF point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of BRAF via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1-8 of the AKAP9 gene and exons 9-18 of BRAF. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The AKAP9-BRAF fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas BRAF point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Proteínas de Ancoragem à Quinase A , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Adulto , Animais , Sequência de Bases , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Acidente Nuclear de Chernobyl , Criança , Chlorocebus aethiops , Cromossomos Humanos Par 7/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Mutação Puntual/genética , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Recombinação Genética/genética , Neoplasias da Glândula Tireoide/patologia , Fatores de Tempo , Valina/genética , Valina/metabolismoRESUMO
A subset of follicular thyroid carcinomas contains a balanced translocation, t(2;3)(q13;p25), that results in fusion of the paired box gene 8 (PAX8) and peroxisome proliferator-activated receptor gamma (PPARG) genes with concomitant expression of a PAX8-PPARgamma fusion protein, PPFP. PPFP is thought to contribute to neoplasia through a mechanism in which it acts as a dominant-negative inhibitor of wild-type PPARgamma. To better understand this type of follicular carcinoma, we generated global gene expression profiles using DNA microarrays of a cohort of follicular carcinomas along with other common thyroid tumors and used the data to derive a gene expression profile characteristic of PPFP-positive tumors. Transient transfection assays using promoters of four genes whose expression was highly associated with the translocation showed that each can be activated by PPFP. PPFP had unique transcriptional activities when compared with PAX8 or PPARgamma, although it had the potential to function in ways qualitatively similar to PAX8 or PPARgamma depending on the promoter and cellular environment. Bioinformatics analyses revealed that genes with increased expression in PPFP-positive follicular carcinomas include known PPAR target genes; genes involved in fatty acid, amino acid, and carbohydrate metabolism; micro-RNA target genes; and genes on chromosome 3p. These results have implications for the neoplastic mechanism of these follicular carcinomas.
Assuntos
Adenocarcinoma Folicular/genética , Perfilação da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , PPAR gama/genética , Fatores de Transcrição Box Pareados/genética , Neoplasias da Glândula Tireoide/genética , Translocação Genética , Adenocarcinoma Folicular/metabolismo , Adenocarcinoma Folicular/patologia , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 3/genética , Biologia Computacional , Humanos , Proteínas de Fusão Oncogênica/biossíntese , Fator de Transcrição PAX8 , PPAR gama/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Análise de Componente Principal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologiaRESUMO
INTRODUCTION: Telomerase repairs the telomeric ends of chromosomes and is active in nearly all malignant cells. Hepatitis C virus (HCV) is known to be oncogenic and potential interactions with the telomerase system require further study. We determined the effects of HCV infection on human telomerase reverse transcriptase (TERT) expression and enzyme activity in primary human hepatocytes and continuous cell lines. RESULTS: Primary human hepatocytes and Huh-7.5 hepatoma cells showed early de novo TERT protein expression 2-4 days after infection and these events coincided with increased TERT promoter activation, TERT mRNA, and telomerase activity. Immunoprecipitation studies demonstrated that NS3-4A protease-helicase, in contrast to core or NS5A, specifically bound to the C-terminal region of TERT through interactions between helicase domain 2 and protease sequences. Increased telomerase activity was noted when NS3-4A was transfected into cells, when added to reconstituted mixtures of TERT and telomerase RNA, and when incubated with high molecular weight telomerase 'holoenzyme' complexes. The NS3-4A catalytic effect on telomerase was inhibited with primuline or danoprevir, agents that are known to inhibit NS3 helicase and protease activities respectively. In HCV infected cells, NS3-4A could be specifically recovered with telomerase holoenzyme complexes in contrast to NS5A or core protein. HCV infection also activated the effector caspase 7 which is known to target TERT. Activation coincided with the appearance of lower molecular weight carboxy-terminal fragment(s) of TERT, chiefly sized at 45 kD, which could be inhibited with pancaspase or caspase 7 inhibitors. CONCLUSIONS: HCV infection induces TERT expression and stimulates telomerase activity in addition to triggering Caspase activity that leads to increased TERT degradation. These activities suggest multiple points whereby the virus can influence neoplasia. The NS3-4A protease-helicase can directly bind to TERT, increase telomerase activity, and thus potentially influence telomere repair and host cell neoplastic behavior.
Assuntos
Hepacivirus/patogenicidade , Telomerase/metabolismo , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Imunofluorescência , Hepatócitos/metabolismo , Humanos , Imunoprecipitação , Regiões Promotoras Genéticas/genética , RNA/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Thyroid cancer poses a significant clinical challenge, and our understanding of its pathogenesis is incomplete. To gain insight into the pathogenesis of papillary thyroid carcinoma, transcriptional profiles of four normal thyroids and 51 papillary carcinomas (PCs) were generated using DNA microarrays. The tumors were genotyped for their common activating mutations: BRAF V600E point mutation, RET/PTC1 and 3 rearrangement and point mutations of KRAS, HRAS and NRAS. Principal component analysis based on the entire expression data set separated the PCs into three groups that were found to reflect tumor morphology and mutational status. By combining expression profiles with mutational status, we defined distinct expression profiles for the BRAF, RET/PTC and RAS mutation groups. Using small numbers of genes, a simple classifier was able to classify correctly the mutational status of all 40 tumors with known mutations. One tumor without a detectable mutation was predicted by the classifier to have a RET/PTC rearrangement and was shown to contain one by fluorescence in situ hybridization analysis. Among the mutation-specific expression signatures were genes whose differential expression was a direct consequence of the mutation, as well as genes involved in a variety of biological processes including immune response and signal transduction. Expression of one mutation-specific differentially expressed gene, TPO, was validated at the protein level using immunohistochemistry and tissue arrays containing an independent set of tumors. The results demonstrate that mutational status is the primary determinant of gene expression variation within these tumors, a finding that may have clinical and diagnostic significance and predicts success for therapies designed to prevent the consequences of these mutations.
Assuntos
Perfilação da Expressão Gênica , Genes ras , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Superfície Celular/genética , Neoplasias da Glândula Tireoide/genética , Sequência de Bases , Primers do DNA , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Iodeto Peroxidase/metabolismo , Proteínas Proto-Oncogênicas c-ret , Receptores Acoplados a Proteínas G , Transcrição GênicaRESUMO
CONTEXT: RET/PTC rearrangements have been reported in papillary thyroid carcinomas with variable frequency in studies that used different detection methods. OBJECTIVE: Our objective was to determine the role of different detection methods and tumor genetic heterogeneity on RET/PTC detection. DESIGN: Sixty-five papillary carcinomas were analyzed for RET/PTC1 and RET/PTC3 using five detection methods: standard-sensitivity RT-PCR, high-sensitivity RT-PCR, real-time LightCycler RT-PCR, Southern blot analysis, and fluorescence in situ hybridization. RESULTS: RET/PTC rearrangements were detected by standard-sensitivity RT-PCR in 14 tumors. High-sensitivity RT-PCR detected RET/PTC in all of these and in 12 additional cases, where the levels of expression corresponded to one to five positive cells. Real-time LightCycler RT-PCR detected RET/PTC in 12 and Southern blot analysis in 11 tumors. By fluorescence in situ hybridization, 14 tumors were positive, including nine cases with 50-86% positive cells and five cases with 17-35% positive cells. Overall, nine (14%) tumors harbored clonal rearrangements, which were present in the majority of tumor cells and detected by all five methods. Five (8%) cases had subclonal rearrangements present in a smaller portion of tumor cells and detected by most methods. Twelve (18%) tumors had nonclonal RET/PTC that were detected only by high-sensitivity RT-PCR. No other mutations were found in tumors harboring clonal RET/PTC, whereas 60% of tumors with subclonal and 42% of tumors with nonclonal RET/PTC harbored additional mutations. CONCLUSIONS: Our data suggest that broad variability in the reported prevalence of RET/PTC rearrangement is at least in part a result of the use of different detection methods and tumor genetic heterogeneity.
Assuntos
Carcinoma Papilar/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Neoplasias da Glândula Tireoide/genética , Southern Blotting , Rearranjo Gênico , Genes ras , Humanos , Hibridização in Situ Fluorescente , Proteínas Proto-Oncogênicas B-raf/genética , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Papillary carcinoma is the most common type of thyroid malignancy. It has been recently shown that these tumors commonly have one of three genetic alterations: BRAF point mutations, RET/PTC rearrangements, or RAS point mutations. In this study, we analyze the relationship between these alterations and the microscopic features of papillary carcinomas, their clinical features, and prognostic characteristics. Ninety-seven papillary carcinomas were studied; in all cases, frozen tissue was available for nucleic acid extraction. Of 96 unselected cases, 42% were positive for BRAF, 18% for RET/PTC, and 15% for RAS mutations. Morphologic features were evaluated in detail in 61 cases and 6 characteristic nuclear features and 3 additional microscopic features were assessed quantitatively. At least 4 nuclear features were found in each tumor, with nuclear pseudoinclusions being the least frequent finding in all mutation groups. BRAF mutations were associated with older patient age, typical papillary appearance or the tall cell variant, a higher rate of extrathyroidal extension, and more advanced tumor stage at presentation. RET/PTC rearrangements presented at younger age and had predominantly typical papillary histology, frequent psammoma bodies, and a high rate of lymph node metastases. Tumors with RAS mutations were exclusively the follicular variant of papillary carcinoma and correlated with significantly less prominent nuclear features and low rate of lymph node metastases. These findings demonstrate that BRAF, RET/PTC, and RAS mutations are associated with distinct microscopic, clinical, and biologic features of thyroid papillary carcinomas.
Assuntos
Carcinoma Papilar/genética , Carcinoma Papilar/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Adulto , Fatores Etários , Feminino , Humanos , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-ret/genéticaRESUMO
Thyroid papillary cancers (PTCs) are associated with activating mutations of genes coding for RET or TRK tyrosine kinase receptors, as well as of RAS genes. Activating mutations of BRAF were reported recently in most melanomas and a small proportion of colorectal tumors. Here we show that a somatic mutation of BRAF, V599E, is the most common genetic change in PTCs (28 of 78; 35.8%). BRAF(V599E) mutations were unique to PTCs, and not found in any of the other types of differentiated follicular neoplasms arising from the same cell type (0 of 46). Moreover, there was no overlap between PTC with RET/PTC, BRAF, or RAS mutations, which altogether were present in 66% of cases. The lack of concordance for these mutations was highly unlikely to be a chance occurrence. Because these signaling proteins function along the same pathway in thyroid cells, this represents a unique paradigm of human tumorigenesis through mutation of three signaling effectors lying in tandem.
Assuntos
Carcinoma Papilar/genética , Proteínas de Drosophila , Mutação , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Neoplasias da Glândula Tireoide/genética , Proteínas ras/fisiologia , Transformação Celular Neoplásica/genética , Éxons , Humanos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/genética , Células Tumorais Cultivadas , Proteínas ras/genéticaRESUMO
betaig-h3 (TGFBI, keratoepithelin) was first identified as a transforming growth factor-beta1 (TGF-beta1)-inducible gene in a human lung adenocarcinoma cell line. It encodes for a secreted extracellular matrix (ECM) protein, which is thought to act on cell attachment and ECM composition. Mutations of the betaig-h3 gene are involved in several corneal dystrophies. Pancreatic cancers display multiple alterations in the TGF-beta signaling pathway and in TGF-beta response genes, such as overexpression of all three TGF-beta isoforms and Smad4 mutations. In this report, we determined that betaig-h3 mRNA levels were induced by TGF-beta1 in two out of five examined pancreatic cancer cell lines (CAPAN-1, PANC-1). In CAPAN-1 cells, which harbor a Smad4 mutation, betaig-h3 but not PAI-1 was induced by TGF-beta1, whereas in PANC-1 cells that express wild-type Smad4, TGF-beta1 induced both PAI-1 and betaig-h3. In human pancreatic tissues, there was a 32.4-fold increase in betaig-h3 mRNA levels in pancreatic cancers in comparison to normal control tissues. In situ hybridization analysis revealed that betaig-h3 mRNA was expressed mainly in the cancer cells within the pancreatic tumor mass. These findings suggest that betaig-h3 is induced by TGF-betas in pancreatic cancer cells even in the presence of Smad4 mutations, which might explain, in part, the increased betaig-h3 mRNA levels observed in pancreatic cancer cells in vivo.
Assuntos
Proteínas da Matriz Extracelular , Proteínas de Neoplasias/biossíntese , Neoplasias Pancreáticas/metabolismo , Adolescente , Adulto , Idoso , Northern Blotting , Clonagem Molecular , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad4 , Transativadores/análise , Transativadores/genética , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1 , Células Tumorais CultivadasRESUMO
Ionizing radiation is a well-known risk factor for thyroid cancer in human populations. Chromosomal rearrangements involving the RET gene, known as RET/PTC, are prevalent in thyroid papillary carcinomas from patients with radiation history. We studied the generation of RET/PTC in HTori-3 immortalized human thyroid cells exposed to a range of doses of gamma-radiation and harvested 2, 5-6, and 9 d later. RET/PTC1 and RET/PTC3 were detected by RT-PCR followed by Southern blotting and hybridization with internal oligonucleotide probes. No RET/PTC was found in cells harvested 2 and 5-6 d after irradiation, whereas 59 RET/PTC events were detected in cells collected 9 d after exposure. The average rate of RET/PTC induction was 0.1 x 10(-6) after exposure to 0.1 Gy, 1.6 x 10(-6) after 1 Gy, 3.0 x 10(-6) after 5 Gy, and 0.9 x 10(-6) after 10 Gy. When adjusted for cell survival, the rate after 10 Gy was comparable with those after 5 Gy. RET/PTC1 was more common than RET/PTC3 after each dose, comprising 80% of all rearrangements. In this study, we demonstrate a dose-dependent induction of RET/PTC rearrangements in human thyroid cells after exposure to 0.1-10 Gy gamma-radiation. This provides additional evidence for a direct link between this genetic event and radiation exposure and offers a powerful experimental system for studying radiation-induced carcinogenesis in the thyroid gland.
Assuntos
Rearranjo Gênico , Neoplasias Induzidas por Radiação/etiologia , Proteínas Oncogênicas/genética , Glândula Tireoide/efeitos da radiação , Neoplasias da Glândula Tireoide/etiologia , Proliferação de Células/efeitos da radiação , Células Cultivadas , Relação Dose-Resposta à Radiação , Raios gama , Humanos , Neoplasias Induzidas por Radiação/genética , Proteínas de Fusão Oncogênica , Proteínas Tirosina Quinases , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genéticaRESUMO
Point mutation of the BRAF gene is a common genetic event in papillary thyroid carcinomas. More recently, it has been found that BRAF can also participate in chromosomal rearrangement. In this study, we explore yet another possible mechanism of BRAF alteration, which involves copy number gain. Using fluorescence in situ hybridization with BRAF specific and chromosome 7 centromeric probes, we studied 62 follicular thyroid tumors and 32 papillary carcinomas. We found that numerical changes in BRAF copy number were rare in papillary thyroid carcinomas, while they occurred in 16-45% of follicular tumors of conventional and oncocytic (Hürthle cell) types. They were due to amplification of the gene or gain of one or more copies of chromosome 7. Tetrasomy for chromosome 7 was overall the most common finding. The changes in BRAF copy number did not overlap with RAS mutations in follicular tumors. In a group of follicular carcinomas, tumors with BRAF copy number gain were significantly more often widely invasive (67%) compared to tumors with no copy number change (18%). By Western blotting, the tumors carrying four copies of the gene revealed higher expression of BRAF protein, suggesting that copy number gain may represent another mechanism of BRAF activation in thyroid tumors.
Assuntos
Adenocarcinoma Folicular/genética , Adenocarcinoma Folicular/patologia , Carcinoma Papilar/genética , Dosagem de Genes , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/genética , Biomarcadores Tumorais/análise , Western Blotting , Carcinoma Papilar/patologia , Cromossomos Humanos Par 7/genética , Análise Mutacional de DNA , Genes ras/genética , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Neoplasias da Glândula Tireoide/patologiaRESUMO
Activating point mutations of the BRAF gene have been recently reported in papillary thyroid carcinomas. In this study, we analyzed 320 thyroid tumors and six anaplastic carcinoma cell lines and detected BRAF mutations in 45 (38%) papillary carcinomas, two (13%) poorly-differentiated carcinomas, three (10%) anaplastic carcinomas, and five (83%) thyroid anaplastic carcinoma cell lines but not in follicular, Hürthle cell, and medullary carcinomas, follicular and Hürthle cell adenomas, or benign hyperplastic nodules. All mutations involved a T-->A transversion at nucleotide 1796. In papillary carcinomas, BRAF mutations were associated with older age, classic papillary carcinoma or tall cell variant histology, extrathyroidal extension, and more frequent presentation at stages III and IV. All BRAF-positive poorly differentiated and anaplastic carcinomas contained areas of preexisting papillary carcinoma, and mutation was present in both the well-differentiated and dedifferentiated components. These data indicate that BRAF mutations are restricted to papillary carcinomas and poorly differentiated and anaplastic carcinomas arising from papillary carcinomas. They are associated with distinct phenotypical and biological properties of papillary carcinomas and may participate in progression to poorly differentiated and anaplastic carcinomas.