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A variety of single-gene human diseases are caused by haploinsufficiency, a genetic condition by which mutational inactivation of one allele leads to reduced protein levels and functional impairment. Translational enhancement of the spare allele could exert a therapeutic effect. Here we developed BOOST, a novel gene-editing approach to rescue haploinsufficiency loci by the change of specific single nucleotides in the Kozak sequence, which controls translation by regulating start codon recognition. We evaluated for translational strength 230 Kozak sequences of annotated human haploinsufficient genes and 4621 derived variants, which can be installed by base editing, by a high-throughput reporter assay. Of these variants, 149 increased the translation of 47 Kozak sequences, demonstrating that a substantial proportion of haploinsufficient genes are controlled by suboptimal Kozak sequences. Validation of 18 variants for 8 genes produced an average enhancement in an expression window compatible with the rescue of the genetic imbalance. Base editing of the NCF1 gene, whose monoallelic loss causes chronic granulomatous disease, resulted in the desired increase of NCF1 (p47phox) protein levels in a relevant cell model. We propose BOOST as a fine-tuned approach to modulate translation, applicable to the correction of dozens of haploinsufficient monogenic disorders independently of the causing mutation.
Assuntos
Haploinsuficiência , Nucleotídeos , Alelos , Códon de Iniciação , Haploinsuficiência/genética , Humanos , RNA Mensageiro/metabolismoRESUMO
Body fluid identification by means of mRNA profiling provides valuable supplementary information in forensic investigations. In particular, the detection of vaginal mucosa mRNA markers is highly relevant in sexual assault cases. Although the vagina undergoes characteristic age-related physiological changes over a lifetime, few studies have evaluated the efficacy of vaginal mRNA markers in women of different ages. In this multicentric study, a 19-plex mRNA profiling assay including vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1) was tested in a collection of 6-20-month-old vaginal swabs obtained from pre- (n = 84) and postmenopausal (n = 55) female volunteer donors. Overall, participating laboratories were able to correctly identify ~85% of samples as vaginal mucosa by mRNA profiling. The assay's success rate did not differ between the two age groups and was not affected by the time interval between swab collection and RNA analysis. MYOZ1 resulted a less sensitive vaginal marker compared to MUC4 and CYP2B7P1. A significant relative increase in the contribution to the total amplification signal was observed for MUC4, compared to CYP2B7P1 and MYOZ1, in postmenopausal women. Observation of other body fluids and tissues different from vaginal mucosa was also evaluated in connection to information on previous sexual activity and menstrual cycle phase at the time of sampling.
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DNA methylation is one of the epigenetic marks which has been studied intensively in recent years for age predicting purposes in the forensic area. In order to integrate age prediction into routine forensic workflow, the purpose of this study was to standardize and optimize a DNA methylation-based protocol tailored to the Italian context. A previously published protocol and age-predictive method was implemented for the analysis of 84 blood samples originating from Central Italy. The study here presented is based on the Single Base Extension method, considering five genes: ELOVL2, FHL2, KLF14, C1orf132, now identified as MIR29B2C, and TRIM59. The precise and specific steps consist of DNA extraction and quantification, bisulfite conversion, amplification of converted DNA, first purification, single base extension, second purification, capillary electrophoresis, and analysis of the results to train and test the tool. The prediction error obtained, expressed as mean absolute deviation, showed a value of 3.12 years in the training set and 3.01 years in the test set. Given that population-based differences in DNA methylation patterns have been previously reported in the literature, it would be useful to further improve the study implementing additional samples representative of the entire Italian population.
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Metilação de DNA , DNA , Projetos Piloto , Ilhas de CpG , Marcadores GenéticosRESUMO
Venous thromboembolism (VTE) is a multifactorial disease determined by a combination of inherited and acquired factors. Inherited factors include mutations in the genes coding for coagulation factors, some of which seem to exert a differential influence on the risk of developing deep vein thrombosis (DVT) and pulmonary embolism (PE). In post-mortem studies of subjects who have died from pulmonary embolism (PE), the analysis of the factors that may have augmented the VTE risk is often limited to acquired factors. This is due to the complexity-and sometimes the unfeasibility-of analyzing genetic factors and to insufficient knowledge of their individual roles in PE development. The present study used formalin-fixed paraffin-embedded (FFPE) tissue to investigate a panel of 12 polymorphisms-the largest ever studied-that affect the VTE risk. Tissue samples came from post-mortem examinations performed by the specialists of the Section of Legal Medicine of the Department of Pathology of Marche's Polytechnic University, and by the specialists of Health Care District Hospital of Imola, on 44 subjects who died from PE in the period 1997-2014. All individuals were found to have at least one mutation affecting the VTE risk. The present study demonstrates that genetic analysis can be performed post-mortem and the results are useful for forensic investigations, especially from MTHFR C677T and PAI-1 4G/5G polymorphisms. Broader studies using the techniques described herein are needed to determine the relative influence of the individual polymorphisms and their interaction in PE deaths.
Assuntos
Predisposição Genética para Doença , Polimorfismo Genético , Embolia Pulmonar/genética , Trombofilia/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Fator V/genética , Fator XIII/genética , Feminino , Fibrinogênio/genética , Formaldeído , Glucuronidase/genética , Heterozigoto , Humanos , Lipoproteínas/genética , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Pessoa de Meia-Idade , Inclusão em Parafina , Inibidor 1 de Ativador de Plasminogênio/genética , Reação em Cadeia da Polimerase , Protrombina/genética , Tromboembolia Venosa/genéticaRESUMO
BACKGROUND: Many recent studies using ChIP-seq approaches cross-referenced to trascriptome data and also to potentially unbiased in vitro DNA binding selection experiments are detailing with increasing precision the p53-directed gene regulatory network that, nevertheless, is still expanding. However, most experiments have been conducted in established cell lines subjected to specific p53-inducing stimuli, both factors potentially biasing the results. RESULTS: We developed p53retriever, a pattern search algorithm that maps p53 response elements (REs) and ranks them according to predicted transactivation potentials in five classes. Besides canonical, full site REs, we developed specific pattern searches for non-canonical half sites and 3/4 sites and show that they can mediate p53-dependent responsiveness of associated coding sequences. Using ENCODE data, we also mapped p53 REs in about 44,000 distant enhancers and identified a 16-fold enrichment for high activity REs within those sites in the comparison with genomic regions near transcriptional start sites (TSS). Predictions from our pattern search were cross-referenced to ChIP-seq, ChIP-exo, expression, and various literature data sources. Based on the mapping of predicted functional REs near TSS, we examined expression changes of thirteen genes as a function of different p53-inducing conditions, providing further evidence for PDE2A, GAS6, E2F7, APOBEC3H, KCTD1, TRIM32, DICER, HRAS, KITLG and TGFA p53-dependent regulation, while MAP2K3, DNAJA1 and potentially YAP1 were identified as new direct p53 target genes. CONCLUSIONS: We provide a comprehensive annotation of canonical and non-canonical p53 REs in the human genome, ranked on predicted transactivation potential. We also establish or corroborate direct p53 transcriptional control of thirteen genes. The entire list of identified and functionally classified p53 REs near all UCSC-annotated genes and within ENCODE mapped enhancer elements is provided. Our approach is distinct from, and complementary to, existing methods designed to identify p53 response elements. p53retriever is available as an R package at: http://tomateba.github.io/p53retriever .
Assuntos
Genoma Humano , Elementos de Resposta/genética , Proteína Supressora de Tumor p53/genética , Algoritmos , Sítios de Ligação , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Bases de Dados Genéticas , Doxorrubicina/farmacologia , Humanos , Imidazóis/farmacologia , Internet , Piperazinas/farmacologia , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Elementos de Resposta/efeitos dos fármacos , Sítio de Iniciação de Transcrição , Ativação Transcricional , Transcriptoma/efeitos dos fármacos , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Interface Usuário-ComputadorRESUMO
A high-throughput matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS)-based method was here developed to genotype 16 high-risk human papillomavirus (HPV) types in cervical cytology specimens. This method was compared to a commercial kit, the Inno-LiPA HPV genotyping assay, which detects a broad spectrum of HPV types. HPV DNA was assessed by the two methods in a total of 325 cervical cytology specimens collected in PreservCyt® solution. The overall agreement was almost perfect (Cohen's k=0.86) in term of positive and negative cases. Indeed, HPV types 16, 35, 56 and 66 showed the highest agreement values (>0.80). The highest agreement values (K >0.80) were found for all 16 HPV types in single infections, but only for HPV 16, 35, 45 and 56 in multiple infections. In conclusion, the high-throughput MS-based method developed here is well-suited for broad spectrum HPV genotyping in large-scale epidemiological studies.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Feminino , Genótipo , Humanos , Papillomaviridae/química , Papillomaviridae/classificação , Papillomaviridae/genéticaRESUMO
BACKGROUND: Sudden cardiac death (SCD) is the clinical outcome of a lethal arrhythmia that can develop on the background of unrecognized channelopathies or cardiomyopathies. Several susceptibility genes have been identified for the congenital forms of these cardiac diseases, including caveolin-3 (Cav-3) gene. In the heart Cav-3 is the main component of caveolae, plasma membrane domains that regulate multiple cellular processes highly relevant for cardiac excitability, such as trafficking, calcium homeostasis, signal transduction and cellular response to injury. Here we characterized a new putative Cav-3 variant, Cav-3 V82I, found in a patient with SCD. RESULTS: In heterologous systems Cav-3 V82I was expressed at significantly higher level than Cav-3 WT and accumulated within the cells. Cells expressing Cav-3 V82I exhibited a decreased activation of extracellular-signal-regulated kinases (ERKs) and were more vulnerable to sub-lethal osmotic stress. CONCLUSION: Considering that abnormal loss of myocytes can play a mechanistic role in lethal cardiac diseases, we suggest that the detrimental effect of Cav-3 V82I variant on cell viability may participate in determining the susceptibility to cardiac death.
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Caveolina 3 , Morte Súbita , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mutação de Sentido Incorreto , Miócitos Cardíacos/metabolismo , Substituição de Aminoácidos , Animais , Caveolina 3/genética , Caveolina 3/metabolismo , Linhagem Celular , Cricetinae , Ativação Enzimática/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Humanos , Masculino , Miócitos Cardíacos/patologiaRESUMO
New therapeutic strategies for glioblastoma treatment, especially tackling the tumour's glioblastoma stem cell (GSC) component, are an urgent medical need. Recently, mitochondrial translation inhibition has been shown to affect GSC growth, clonogenicity, and self-renewal capability, therefore becoming an attractive therapeutic target. The combination of streptogramins B and A antibiotics quinupristin/dalfopristin (Q/D), which inhibits mitochondrial ribosome function, affects GSCs more effectively in vitro than the standard of care temozolomide. Here, docking calculations based on the cryo-EM structure of the Q/D-bound mitochondrial ribosome have been used to develop a series of streptogramin A derivatives. We obtained twenty-two new and known molecules starting from the dalfopristin and virginiamycin M1 scaffolds. A structure-activity relationship refinement was performed to evaluate the capability of these compounds to suppress GSC growth and inhibit mitochondrial translation, either alone or in combination with quinupristin. Finally, quantitative ultra HPLC-mass spectrometry allowed us to assess the cell penetration of some of these derivatives. Among all, the fluorine derivatives of dalfopristin and virginiamycin M1, (16R)-1e and (16R)-2e, respectively, and flopristin resulted in being more potent than the corresponding lead compounds and penetrating to a greater extent into the cells. We, therefore, propose these three compounds for further evaluation in vivo as antineoplastic agents.
Assuntos
Glioblastoma , Estreptograminas , Humanos , Estreptogramina A , Glioblastoma/tratamento farmacológico , Antibacterianos/química , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas , Testes de Sensibilidade MicrobianaRESUMO
The use of rapid-acting insulin analogs as routes of administration other than IV has never been described for the treatment of dogs with diabetic ketoacidosis (DKA). This study aims to compare the efficacy and safety of a new protocol based on IM administration of insulin lispro with that of low-dose IV continuous rate infusion of regular insulin in the treatment of canine DKA. Client-owned dogs with naturally occurring DKA were included. Dogs treated with IM insulin lispro (Group L, n = 11) received 0.25 U/kg. The goal was to achieve a drop of at least 10% in blood glucose between 1 h and the next. If this goal was not achieved, the insulin dose was repeated hourly; otherwise, the insulin dose was not repeated up to a maximum of 3 h, after which the insulin dose was repeated anyway. When blood glucose was ≤250 mg/dL, the insulin dose was reduced to 0.125 U/kg IM every 3 h. Cases receiving IV continuous rate infusion of regular insulin (Group R, n = 13) were treated according to a previously published protocol. The median time to resolution of ketosis was significantly shorter in Group L (12 h; range, 4-27 h) compared to Group R (23 h; 10-46 h; P = 0.04). The median times to resolution of acidemia and ketoacidosis were 13 h (4-35 h) and 17.5 h (4-35 h) in Group L, and 22 h (9-80 h) and 23.5 h (10-80 h) in Group R, respectively. These differences were not significant (P = 0.06 and P = 0.09, respectively). The median length of hospitalization did not differ significantly between groups (P = 0.67). There were no differences in the frequency and severity of adverse events (hypoglycemia, hypokaliemia, and hypophosphatemia) between groups. The new protocol based on IM administration of insulin lispro preliminarily appears effective and safe for treatment of canine DKA.
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Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer's instructions.
Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase/métodos , Sequenciamento Completo do Genoma/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Betacoronavirus/patogenicidade , COVID-19 , Chlorocebus aethiops , Primers do DNA/normas , Feminino , Genoma Viral , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Reação em Cadeia da Polimerase/normas , SARS-CoV-2 , Células Vero , Sequenciamento Completo do Genoma/normasRESUMO
MiR-222 and miR-126 are associated with asbestos exposure and the ensuing malignancy, but the mechanism(s) of their regulation remain unclear. We evaluated the mechanism by which asbestos regulates miR-222 and miR-126 expression in the context of cancer etiology. An 'in vitro' model of carcinogen-induced cell transformation was used based on exposing bronchial epithelium BEAS-2B cells to three different carcinogens including asbestos. Involvement of the EGFR pathway and the role of epigenetics have been investigated in carcinogen-transformed cells and in malignant mesothelioma, a neoplastic disease associated with asbestos exposure. Increased expression of miR-222 and miR-126 were found in asbestos-transformed cells, but not in cells exposed to arsenic and chrome. Asbestos-mediated activation of the EGFR pathway and macrophages-induced inflammation resulted in miR-222 upregulation, which was reversed by EGFR inhibition. Conversely, asbestos-induced miR-126 expression was affected neither by EGFR modulation nor inflammation. Rather than methylation of the miR-126 host gene EGFL7, epigenetic mechanism involving DNMT1- and PARP1-mediated chromatin remodeling was found to upregulate of miR-126 in asbestos-exposed cells, while miR-126 was downregulated in malignant cells. Analysis of MM tissue supported the role of PARP1 in miR-126 regulation. Therefore, activation of the EGFR pathway and the PARP1-mediated epigenetic regulation both play a role in asbestos-induced miRNA expression, associated with in asbestos-induced carcinogenesis and tumor progression.
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Amianto/efeitos adversos , Carcinógenos/química , Neoplasias Pulmonares/genética , Mesotelioma/genética , MicroRNAs/metabolismo , Idoso , Humanos , Neoplasias Pulmonares/patologia , Masculino , Mesotelioma/patologia , Mesotelioma MalignoRESUMO
We present a new model of ESR1 network regulation based on analysis of Doxorubicin, Estradiol, and TNFα combination treatment in MCF-7. We used Doxorubicin as a therapeutic agent, TNFα as marker and mediator of an inflammatory microenvironment and 17ß-Estradiol (E2) as an agonist of Estrogen Receptors, known predisposing factor for hormone-driven breast cancer, whose pharmacological inhibition reduces the risk of breast cancer recurrence. Based on the results of transcriptomics analysis, we found 71 differentially expressed genes that are specific for the combination treatment with Doxorubicin + Estradiol + TNFα in comparison with single or double treatments. The responsiveness to the triple treatment was examined for seven genes by qPCR, of which six were validated, and then extended to four additional cell lines differing for p53 and/or ER status. The results of differential regulation enrichment analysis highlight the role of the ESR1 network that included 36 of 71 specific differentially expressed genes. We propose that the combined activation of p53 and NF-kB transcription factors significantly influences ligand-dependent, ER-driven transcriptional responses, also of the ESR1 gene itself. These results provide a model of coordinated interaction of TFs to explain the Doxorubicin, E2 and TNFα induced repression mechanisms.
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Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/uso terapêutico , Estradiol/uso terapêutico , Fator de Necrose Tumoral alfa/uso terapêutico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Modelos Biológicos , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
MiR-126 has been shown to suppress malignant mesothelioma (MM) by targeting cancer-related genes without inducing toxicity or histopathological changes. Exosomes provide the opportunity to deliver therapeutic cargo to cancer stroma. Here, a tumour stromal model composed of endothelial cells (HUVECs), fibroblasts (IMR-90â¯cells), non-malignant mesothelial cells (Met-5A cells) and MM cells (H28 and MM-B1 cells) was used. The cells were treated with exosomes from HUVECs carrying endogenous (exo-HUVEC) and enriched miR-126 (exo-HUVECmiR-126), and the uptake/turnover of exosomes; miR-126 distribution within the stroma; and effect of miR-126 on cell signalling, angiogenesis and cell proliferation were evaluated. Based on the sensitivity of MM cells to exo-HUVEC miR-126 treatment, miR-126 was distributed differently across stromal cells. The reduced miR-126 content in fibroblasts in favour of endothelial cells reduced angiogenesis and suppressed cell growth in an miR-126-sensitive environment. Conversely, the accumulation of miR-126 in fibroblasts and the reduced level of miR-126 in endothelial cells induced tube formation in an miR-126-resistant environment via VEGF/EGFL7 upregulation and IRS1-mediated cell proliferation. These findings suggest that transfer of miR-126 via HUVEC-derived exosomes represents a novel strategy to inhibit angiogenesis and cell growth in MM.
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Comunicação Celular/fisiologia , Exossomos/metabolismo , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , MicroRNAs/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Carcinogênese/metabolismo , Células Cultivadas , Família de Proteínas EGF/metabolismo , Fibroblastos/metabolismo , Humanos , Mesotelioma Maligno , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Standard serological tests (both EIA and Immunoblotting) have reached high levels of sensitivity and reproducibility, but do not indicate whether infection is recent or longstanding. Since many patients with HIV-1 infection are not usually diagnosed until symptom presentation, the possibility to distinguish between acute and chronic infection has become increasingly important for the purposes of therapeutic decision-making, partner notification and epidemiological surveillance. We evaluated a guanidine-based-antibody-avidity assay in a selected group of recent (within six months from seroconversion) and chronic (more than forty eight months) HIV-1 infections in an attempt to shed more light on the significance of the avidity index in establishing the time of infection. Sera from newly infected individuals showed a low mean avidity index (ranging from 0.35 to 0.60 with a standard deviation 0.09) at baseline and a clear increasing value at the following times of observation. Our data showed that an avidity index <0.70 might be presumptive of infection occurring within 9 months. Avidity index levels might distinguish between acute and chronic infection. The method is semi-automated, inexpensive and easy to perform, and estimates the time elapsed from seroconversion, thereby identifying a recent infection.
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Afinidade de Anticorpos , Anticorpos Anti-HIV/sangue , Infecções por HIV/diagnóstico , HIV-1/imunologia , Doença Aguda , Adulto , Feminino , Infecções por HIV/virologia , Soropositividade para HIV , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The analysis of microorganism population is crucial in several medical fields. This is especially true in legal and occupational medicine, where the specialist can be asked to perform an evaluation of several environmental matrices. In these two medical fields an accurate microbiological analysis is part of a wide process aimed to the definition of the interactions between human beings and environment. In legal medicine it is important to deserve attention to the identification of microbiological traces in order to better understand past events, while in occupational and preventive medicine the microbiological evaluation of environmental samples is crucial for an effective risk management and the definition of safety procedures. The achievement of these objectives requires the comprehension of microbial biodiversity and not only the identification of few biomarkers. In the present paper, the complexity of this process is highlighted through the presentation of typical scenarios where microorganism population analyses are relevant in legal medicine and occupational medicine. The similarities between the microbiological approach in legal and occupational medicine lead to the sharing of laboratory approaches. A description of technological evolution shows how new protocols and procedures are supporting a wider microbiological comprehension of specimens. The development of molecular tools has opened new opportunities, but it has underlined the need for the implementation of new standardized procedures dedicated to these medical fields, where science and medicine interact with the law. In addition, the rapid evolution of massive parallel sequencing technologies requires the implementation of new bioinformatic tools with a user-friendly interface.
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Microbiologia Ambiental , Medicina Legal , Medicina do Trabalho , HumanosRESUMO
Breast cancer treatment often includes Doxorubicin as adjuvant as well as neoadjuvant chemotherapy. Despite its cytotoxicity, cells can develop drug resistance to Doxorubicin. Uncovering pathways and mechanisms involved in drug resistance is an urgent and critical aim for breast cancer research oriented to improve treatment efficacy. Here we show that Doxorubicin and other chemotherapeutic drugs induce the expression of ETV7, a transcriptional repressor member of ETS family of transcription factors. The ETV7 expression led to DNAJC15 down-regulation, a co-chaperone protein whose low expression was previously associated with drug resistance in breast and ovarian cancer. There was a corresponding reduction in Doxorubicin sensitivity of MCF7 and MDA-MB-231 breast cancer cells. We identified the binding site for ETV7 within DNAJC15 promoter and we also found that DNA methylation may be a factor in ETV7-mediated DNAJC15 transcriptional repression. These findings of an inverse correlation between ETV7 and DNAJC15 expression in MCF7 cells in terms of Doxorubicin resistance, correlated well with treatment responses of breast cancer patients with recurrent disease, based on our analyses of reported genome-wide expression arrays. Moreover, we demonstrated that ETV7-mediated Doxorubicin-resistance involves increased Doxorubicin efflux via nuclear pumps, which could be rescued in part by DNAJC15 up-regulation. With this study, we propose a novel role for ETV7 in breast cancer, and we identify DNAJC15 as a new target gene responsible for ETV7-mediated Doxorubicin-resistance. A better understanding of the opposing impacts of Doxorubicin could improve the design of combinatorial adjuvant regimens with the aim of avoiding resistance and relapse.
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Neoplasias da Mama/genética , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas Proto-Oncogênicas c-ets/genética , Células A549 , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Recidiva Local de Neoplasia/genéticaRESUMO
AIM: HPV DNA has never been investigated in nipple discharges (ND) and serum-derived extracellular vesicles, although its presence has been reported in ductal lavage fluids and blood specimens. MATERIALS & METHODS: We analyzed 50 ND, 22 serum-derived extracellular vesicles as well as 51 pathologic breast tissues for the presence of 16 HPV DNA types. RESULTS: We show that the presence of HPV DNA in the ND is predictive of HPV DNA-positive breast lesions and that HPV DNA is more represented in intraductal papillomas. We also show the presence of HPV DNA in the serum-derived extracellular vesicles. CONCLUSION: Our data supports the use of liquid biopsy to detect HPV DNA in breast pathology.
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Mama/patologia , DNA Viral/metabolismo , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Vesículas Extracelulares/química , Feminino , Humanos , Biópsia Líquida , Pessoa de Meia-Idade , Papiloma Intraductal/patologia , Papillomaviridae/genética , Adulto JovemRESUMO
BACKGROUND: Since HIV infection cannot be completely eliminated due to the establishment of latently infected CD4+ T cell reservoirs, there is an urgent need for a clearer understanding of comparative resistance profiles between plasma and PBMC in HIV-1 patients. OBJECTIVES: To assess the prevalence of mutations associated with drug resistance and to compare cell free and cell-associated strains. STUDY DESIGN: Genotypic resistance analysis on viral DNA and plasma was performed in 31 therapy naive patients with chronic infection to check reverse transcriptase (RT) and protease resistance associated mutations before beginning antiretroviral therapy. RESULTS: Direct sequencing of DNA provirus disclosed key mutations (such as G190A/S, V106A, K103N and T215F) to RT inhibitors more frequently (7 patients out 31) than in plasma RNA (2 out of 31). In addition, major mutations (D30N, M46I, I50V, I84V) associated with drug resistance in the PR region were only found in PBMCs. CONCLUSIONS: Despite the small number of patients, our results show a different resistance profile between plasma and PBMC compartments and may yield additional information for first-line antiretroviral regimens. Further investigations on larger series followed-up for a longer period of time are required to obtain in-depth information on the meaning of the mutations detectable in plasma and/or in PBMCs.
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Farmacorresistência Viral/genética , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/efeitos dos fármacos , Linfócitos/virologia , Plasma/virologia , Adulto , Substituição de Aminoácidos/genética , DNA Viral/genética , Feminino , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/genética , Humanos , Masculino , Mutação/genética , Filogenia , RNA Viral/genética , Análise de Sequência de DNARESUMO
Genotypic resistance analysis on viral DNA and plasma was performed in 56 therapy naive patients with recent and chronic infection to assess the prevalence of mutations associated with drug resistance and compare cell-free and cell-associated strains. Direct sequencing of DNA provirus disclosed key mutations to RT inhibitors more frequently than in plasma RNA. In addition, major mutations associated with drug resistance in the PR region were only found in PBMCs. Although our data are limited to a small cohort, they show a different resistance profile between plasma and PBMC compartments and may yield additional information for first-line antiretroviral regimens.
Assuntos
Infecções por HIV/virologia , HIV-1/genética , Leucócitos Mononucleares/virologia , Plasma/virologia , Provírus/genética , Fármacos Anti-HIV/farmacologia , Terapia Antirretroviral de Alta Atividade , DNA Viral/genética , Farmacorresistência Viral/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Humanos , Mutação , RNA Viral/genética , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
Lysosomes are important cytoplasmic organelles whose critical functions in cells are increasingly being understood. In particular, despite the long-standing accepted concept about the role of lysosomes as cellular machineries solely assigned to degradation, it has been demonstrated that they play active roles in homeostasis and even in cancer biology. Indeed, it is now well documented that during the process of cellular transformation and cancer progression lysosomes are changing localization, composition, and volume and, through the release of their enzymes, lysosomes can also enhance cancer aggressiveness. LAMPs (lysosome associated membrane proteins) represent a family of glycosylated proteins present predominantly on the membrane of lysosomes whose expression can vary among different tissues, suggesting a separation of functions. In this review we focus on the functions and roles of the different LAMP family members, with a particular emphasis on cancer progression and metastatic spread. LAMP proteins are involved in many different aspects of cell biology and can influence cellular processes such as phagocytosis, autophagy, lipid transport, and aging. Interestingly, for all the five members identified so far (LAMP1, LAMP2, LAMP3, CD68/Macrosialin/LAMP4, and BAD-LAMP/LAMP5), a role in cancer has been suggested. While this is well documented for LAMP1 and LAMP2, the involvement of the other three proteins in cancer progression and aggressiveness has recently been proposed and remains to be elucidated. Here we present different examples about how LAMP proteins can influence and support tumor growth and metastatic spread, emphasizing the impact of each single member of the family.