RESUMEN
An amino acid sequence variant (SV) is defined as an unintended amino acid substitution in protein drug products. SVs contribute to product heterogeneity and can potentially impact product quality, safety, immunogenicity, and efficacy. The analysis of biotherapeutics for SVs is important throughout the product life cycle including clone selection, development of nutrient feed strategies, commercial manufacturing process, and post-approval changes to monitor product quality. The proposed analytical procedure for SVs consists of both qualitative (identification of SVs) and quantitative (quantitation of identified SVs) components. The complexities of SV analysis and the variety of current procedures highlight the need for a systematic approach for assessing the capability of these methodologies to reliably identify and quantitate SVs in biotherapeutics. We described here a "spike-control" approach for evaluating SV analytical procedure. The concept was adopted from quality control samples routinely used in analytical procedure validation. One FDA approved monoclonal antibody (mAb) was spiked with accurate amounts of highly homologous mAb to create mAb samples containing low yet accurate levels of "artificial" SVs. Spike-control samples were denatured, reduced, alkylated, digested and then analyzed by high resolution Orbitrap mass spectrometry. In silico analysis revealed four single amino acid differences between the two mAbs that could be used to represent SVs in the spike-control samples. All four "artificial" SVs were reliably identified by the current workflow. Analytical range (0.01% to 2%), accuracy and precision of identified SVs have also been evaluated. Overall, spike-control sample(s) helped to demonstrate that the SV analytical procedure (i.e., sample preparation, LC separation, mass spectrometry determinations and bioinformatic software) was fit for purpose and suitable for the identification and quantitation of SVs at a pre-determined threshold.
Asunto(s)
Anticuerpos Monoclonales , Programas Informáticos , Anticuerpos Monoclonales/química , Secuencia de Aminoácidos , Espectrometría de Masas/métodosRESUMEN
Recent advances in high resolution mass spectrometry (MS) instrumentation and semi-automated software have led to a push toward the use of MS-based methods for quality control (QC) testing of therapeutic proteins in a cGMP environment. The approach that is most commonly being proposed for this purpose is known as the multi-attribute method (MAM). MAM is a promising approach that provides some distinct benefits compared to conventional methods currently used for QC testing of protein therapeutics, such as CEX, HILIC, and CE-SDS. Because MS-based methods have not been regularly used in this context in the past, new scientific and regulatory questions should be addressed prior to the final stages of implementation. We have categorized these questions into four major aspects for MAM implementation in a cGMP environment for both new and existing products: risk assessment, method validation, capabilities and specificities of the New Peak Detection (NPD) feature, and comparisons to conventional methods. This perspective outlines considerations for each of these main points and suggests approaches to help address potential issues.
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Cromatografía Liquida/métodos , Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Péptidos/análisis , Proteínas/química , Animales , Anticuerpos Monoclonales/química , Humanos , Control de CalidadRESUMEN
Evolving immunogenicity assay performance expectations and a lack of harmonized neutralizing antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. A team of experts within the American Association of Pharmaceutical Scientists' Therapeutic Product Immunogenicity Community across industry and the Food and Drug Administration addressed challenges unique to cell-based and non-cell-based neutralizing antibody assays. Harmonization of validation expectations and data reporting will facilitate filings to health authorities and are described in this manuscript. This team provides validation testing and reporting strategies and tools for the following assessments: (1) format selection; (2) cut point; (3) assay acceptance criteria; (4) control precision; (5) sensitivity including positive control selection and performance tracking; (6) negative control selection; (7) selectivity/specificity including matrix interference, hemolysis, lipemia, bilirubin, concomitant medications, and structurally similar analytes; (8) drug tolerance; (9) target tolerance; (10) sample stability; and (11) assay robustness.
Asunto(s)
Anticuerpos Neutralizantes , Preparaciones Farmacéuticas , Tolerancia a MedicamentosRESUMEN
Breast cancer is the most common tumor among women with inherited mutations in the p53 gene (Li-Fraumeni syndrome). The tumors represent the basal-like subtype, which has been suggested to originate from mammary stem/progenitor cells. In mouse mammary epithelium, mammosphere-forming potential was increased with decreased dosage of the gene encoding the p53 tumor suppressor protein (Trp53). Limiting dilution transplantation also showed a 3.3-fold increase in the frequency of long-term regenerative mammary stem cells in Trp53-/- mice. The repression of mammospheres by p53 was apparent despite the absence of apoptotic responses to radiation indicating a dissociation of these two activities of p53. The effects of p53 on progenitor cells were also observed in TM40A cells using both mammosphere-forming assays and the DsRed-let7c-sensor. The frequency of long-term label-retaining epithelial cells was decreased in Trp53-/- mammary glands indicating that asymmetric segregation of DNA is diminished and contributes to the expansion of the mammary stem cells. Treatment with an inhibitor of γ-secretase (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) reduced the number of Trp53-/- mammospheres to the level found in Trp53+/+ cells. These results demonstrate that basal levels of p53 restrict mammary stem/progenitor cells through Notch and that the Notch pathway is a therapeutic target to prevent expansion of this vulnerable pool of cells.
Asunto(s)
Apoptosis , Genes p53/fisiología , Glándulas Mamarias Humanas/citología , Receptores Notch/metabolismo , Células Madre/fisiología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/prevención & control , Células Cultivadas , Dipéptidos/farmacología , Células Epiteliales/fisiología , Femenino , Genes p53/genética , Humanos , Síndrome de Li-Fraumeni/genética , Síndrome de Li-Fraumeni/patología , Síndrome de Li-Fraumeni/terapia , Glándulas Mamarias Humanas/efectos de los fármacos , Glándulas Mamarias Humanas/metabolismo , Ratones , Ratones Endogámicos BALB C , Receptores Notch/antagonistas & inhibidores , Trasplante de Células Madre , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication.
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Anticuerpos , Anticuerpos NeutralizantesRESUMEN
The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.
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Receptores Quiméricos de Antígenos , Vacunas , Biomarcadores/análisis , Sistemas CRISPR-Cas , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Inmunoterapia Activa , Reacción en Cadena de la PolimerasaRESUMEN
Mutation and loss of function in p53 are common features among human breast cancers. Here we use BALB/c-Trp53+/- mice as a model to examine the sequence of events leading to mammary tumors. Mammary gland proliferation rates were similar in both BALB/c-Trp53+/- mice and wild-type controls. In addition, sporadic mammary hyperplasias were rare in BALB/c-Trp53+/- mice and not detectably different from those of wild-type controls. Among the 28 mammary tumors collected from BALB/c-Trp53+/- mice, loss of heterozygosity for Trp53 was detected in more than 90% of invasive mammary tumors. Transplantation of Trp53+/- ductal hyperplasias also indicated an association between loss of the wild-type allele of Trp53 and progression to invasive carcinomas. Therefore, loss of p53 function seems to be a rate-limiting step in progression. Moreover, expression of biomarkers such as estrogen receptor alpha, progesterone receptor, Her2/Neu, and activated Notch1 varied among mammary tumors, suggesting that multiple oncogenic lesions collaborate with loss of p53 function. Expression of biomarkers was retained when tumor fragments were transplanted to syngeneic hosts. Tumors expressing solely luminal or basal keratins were also observed (27 and 11%, respectively), but the largest class of tumors expressed both luminal and basal keratins (62%). Overall, this panel of transplantable tumors provides a resource for detailed evaluation of the cell lineages undergoing transformation and preclinical testing of therapeutic agents targeting a variety of oncogenic pathways including cancer stem cells.
Asunto(s)
Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Lesiones Precancerosas/patología , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Animales , Femenino , Regulación Neoplásica de la Expresión Génica , Queratinas/metabolismo , Pérdida de Heterocigocidad/genética , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Lesiones Precancerosas/genética , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Receptores Notch/metabolismo , Receptores de Progesterona/metabolismoRESUMEN
Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting.
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Anticuerpos , Bioensayo , Europa (Continente) , Estados UnidosRESUMEN
The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity). Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation) and Part 2B (Regulatory Input) are published in volume 13 of Bioanalysis, issues 4 and 5 (2020), respectively.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Citometría de Flujo , Terapia Genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Vacunas/análisis , Humanos , Control de Calidad , Receptores Quiméricos de Antígenos/análisis , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Estrogen and progestins are essential for mammary growth and differentiation but also enhance the activity of the p53 tumor suppressor protein in the mammary epithelium. However, the pathways by which these hormones regulate p53 activity are unknown. Microarrays were used to profile the transcriptional changes within the mammary gland after administration of either vehicle, 17beta-estradiol (E), or progesterone (P) individually and combined (EP). Treatment with EP yielded 1182 unique genes that were differentially expressed compared to the vehicle-treated group. Although 30% of genes were responsive to either E or P individually, combined treatment with both EP had a synergistic effect accounting for 60% of the differentially regulated genes. Analysis of protein-protein interactions identified p53, RelA, Snw1, and Igfals as common targets of genes regulated by EP. RelA and p53 form hubs within a network connected by genes that are regulated by EP and that may coordinate the competing functions of RelA and p53 in proliferation and survival of cells. Induction of early growth response 1 (Egr1) and Stratifin (Sfn) (also known as 14-3-3sigma) by EP was confirmed by reverse transcription-quantitative PCR and shown to be p53 independent. In luciferase reporter assays, Egr1 was shown to enhance transcriptional activation by p53 and inhibit nuclear factor kappaB activity. These results identify a gene expression network that provides redundant activation of RelA to support proliferation as well as sensitize p53 to ensure proper surveillance and integration of their competing functions through factors such as Egr1, which both enhance p53 and inhibit RelA.
Asunto(s)
Estradiol/farmacología , Glándulas Mamarias Animales/fisiología , Progesterona/farmacología , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas 14-3-3/genética , Animales , Neoplasias de la Mama , Línea Celular Transformada , Línea Celular Tumoral , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Epitelio/efectos de los fármacos , Epitelio/fisiología , Femenino , Perfilación de la Expresión Génica , Humanos , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Ovariectomía , Transcripción Genética/fisiologíaRESUMEN
The hypothesis that cancer stem cells are responsible for the chemoresistant and metastatic phenotypes of many breast cancers has gained support using cell-sorting strategies to enrich the tumor-initiating population of cells. The mechanisms regulating the cancer stem cell pool, however, are less clear. Two recent publications suggest that loss of p53 permits expansion of presumptive cancer stem cells in mouse mammary tumors and in human breast cell lines. These results add restriction of cancer stem cells as a new tumor suppressor activity attributed to p53.
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Regulación Neoplásica de la Expresión Génica , Genes p53 , Células Madre Neoplásicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Mutación , FenotipoRESUMEN
INTRODUCTION: Treatment with estrogen and progesterone (E+P) mimics the protective effect of parity on mammary tumors in rodents and depends upon the activity of p53. The following experiments tested whether exogenous E+P primes p53 to be more responsive to DNA damage and whether these pathways confer resistance to mammary tumors in a mouse model of Li-Fraumeni syndrome. METHODS: Mice that differ in p53 status (Trp53+/+, Trp53+/-, Trp53-/-) were treated with E+P for 14 days and then were tested for p53-dependent responses to ionizing radiation. Responses were also examined in parous and age-matched virgins. The effects of hormonal exposures on tumor incidence were examined in BALB/c-Trp53+/- mammary tissues. RESULTS: Nuclear accumulation of p53 and apoptotic responses were increased similarly in the mammary epithelium from E+P-treated and parous mice compared with placebo and age-matched virgins. This effect was sustained for at least 7 weeks after E+P treatment and did not depend on the continued presence of ovarian hormones. Hormone stimulation also enhanced apoptotic responses to ionizing radiation in BALB/c-Trp53+/- mice but these responses were intermediate compared with Trp53+/+ and Trp-/- tissues, indicating haploinsufficiency. The appearance of spontaneous mammary tumors was delayed by parity in BALB/c-Trp53+/- mice. The majority of tumors lacked estrogen receptor (ER), but ER+ tumors were observed in both nulliparous and parous mice. However, apoptotic responses to ionizing radiation and tumor incidence did not differ among outgrowths of epithelial transplants from E+P-treated donors and nulliparous donors. CONCLUSION: Therefore, E+P and parity confer a sustained increase in p53-mediated apoptosis within the mammary epithelium and suppress mammary tumorigenesis, but this effect was not retained in epithelial outgrowths.
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Apoptosis , Estrógenos/metabolismo , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/genética , Progesterona/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/fisiología , Animales , Daño del ADN , Modelos Animales de Enfermedad , Femenino , Síndrome de Li-Fraumeni/terapia , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Placebos , Receptores de Estrógenos/metabolismoRESUMEN
The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies' input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of Bioanalysis, issues 22 and 24 (2018), respectively.
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Antígenos/análisis , Bioensayo/normas , Biomarcadores/análisis , Legislación Médica/tendencias , Estados UnidosRESUMEN
Cytochrome c adsorbed to anionic nanoparticles is selectively proteolyzed by trypsin, providing a mechanism for the catalytic degradation of proteins.
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Citocromos c/química , Electroforesis en Gel de Agar/métodos , Nanopartículas/química , Tripsina/química , Adsorción , Sitios de Unión , Catálisis , Sensibilidad y Especificidad , Propiedades de Superficie , Factores de TiempoRESUMEN
Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2-positive metastatic breast cancer. It consists of trastuzumab, a humanized mAb directed against HER2, and a microtubule inhibitor, DM1, conjugated to trastuzumab via a thioether linker. Hepatotoxicity is one of the serious adverse events associated with T-DM1 therapy. Mechanisms underlying T-DM1-induced hepatotoxicity remain elusive. Here, we use hepatocytes and mouse models to investigate the mechanisms of T-DM1-induced hepatotoxicity. We show that T-DM1 is internalized upon binding to cell surface HER2 and is colocalized with LAMP1, resulting in DM1-associated cytotoxicity, including disorganized microtubules, nuclear fragmentation/multiple nuclei, and cell growth inhibition. We further demonstrate that T-DM1 treatment significantly increases the serum levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in mice and induces inflammation and necrosis in liver tissues, and that T-DM1-induced hepatotoxicity is dose dependent. Moreover, the gene expression of TNFα in liver tissues is significantly increased in mice treated with T-DM1 as compared with those treated with trastuzumab or vehicle. We propose that T-DM1-induced upregulation of TNFα enhances the liver injury that may be initially caused by DM1-mediated intracellular damage. Our proposal is underscored by the fact that T-DM1 induces the outer mitochondrial membrane rupture, a typical morphologic change in the mitochondrial-dependent apoptosis, and mitochondrial membrane potential dysfunction. Our work provides mechanistic insights into T-DM1-induced hepatotoxicity, which may yield novel strategies to manage liver injury induced by T-DM1 or other ADCs.
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Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Maitansina/análogos & derivados , Receptor ErbB-2/metabolismo , Ado-Trastuzumab Emtansina , Animales , Anticuerpos Monoclonales Humanizados/efectos adversos , Antineoplásicos/efectos adversos , Biomarcadores , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Expresión Génica , Humanos , Inmunohistoquímica , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Maitansina/efectos adversos , Maitansina/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Microtúbulos/metabolismo , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/patología , Membranas Mitocondriales/ultraestructura , Modelos Animales , Necrosis/tratamiento farmacológico , Necrosis/patología , Receptor ErbB-2/genética , Trastuzumab , Moduladores de Tubulina/farmacologíaRESUMEN
Cre-mediated cassette exchange has been developed to perform site-specific chromosomal integration using Cre recombinase. Here, site-specific integration with inverted Lox sites was used to investigate the erythroid cis-acting DNA element in specific chromatin contexts in mouse erythroleukemia cells. Single hygromycin-resistant clones were obtained from the selective semi-solid medium containing hygromycin post-electroporation. PCR and Southern blotting analysis showed single-copy integration of target vector in clones A, B and D. Site-specific cassette exchange was performed in clone A with exchange vector and Cre expression plasmid, followed by gancyclovir selection. Flow cytometry was used for analysis of EGFP gene expression. A 732-bp fragment of human beta-globin gene cluster 5' DNase I hypersensitive site 2 (HS2) was exchanged and integrated into clone A in an anti-genomic orientation. The low EGFP expression in clone A-HS may be due to the orientation-dependent gene silencing caused by integration of HS2 in a non-permissive orientation.
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Integrasas/metabolismo , Recombinación Genética , Proteínas Virales/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular Tumoral , Desoxirribonucleasa I/metabolismo , Dimetilsulfóxido/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/genética , Globinas/genética , Globinas/metabolismo , Proteínas Fluorescentes Verdes , Hemina/farmacología , Humanos , Integrasas/genética , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/genéticaRESUMEN
Basal-like triple-negative breast cancers (TNBCs) have poor prognosis. To identify basal-like TNBC dependencies, a genome-wide siRNA lethality screen compared two human breast epithelial cell lines transformed with the same genes: basal-like BPLER and myoepithelial HMLER. Expression of the screen's 154 BPLER dependency genes correlated with poor prognosis in breast, but not lung or colon, cancer. Proteasome genes were overrepresented hits. Basal-like TNBC lines were selectively sensitive to proteasome inhibitor drugs relative to normal epithelial, luminal, and mesenchymal TNBC lines. Proteasome inhibition reduced growth of established basal-like TNBC tumors in mice and blocked tumor-initiating cell function and macrometastasis. Proteasome addiction in basal-like TNBCs was mediated by NOXA and linked to MCL-1 dependence.
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Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Complejo de la Endopetidasa Proteasomal/genética , ARN Interferente Pequeño/análisis , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Pronóstico , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Thermotherapy is a promising technique for the minimally invasive elimination of solid tumors. Here we report the fabrication of protein-coated iron oxide NPs (12 nm core) for use as thermal therapeutic agents. These albumin-passivated NPs are stable under physiological conditions, with rapid heating and cell killing capacity upon alternating magnetic field (AMF) exposure. The mode of action is specific: no measurable cytotoxicity was observed for the particle without AMF or for AMF exposure without the particle.