The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on 19-23 June 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2023 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 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 2) covers the recommendations on Biomarkers, IVD/CDx, LBA and Cell-Based Assays. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 9 and 7 (2024), respectively.
Biomarkers , Cell- and Tissue-Based Therapy , Vaccines , Humans , Biomarkers/analysis , Vaccines/immunology , Flow Cytometry , Biological Assay/methods , European Union , White
Comparative pharmacokinetics (PK) studies have efficiently served as the bridge between autoinjectors and prefilled syringes given the underlying principles that comparable exposure could translate to comparable efficacy and safety. This article discusses approaches used to address uncertainties associated with the observation of noncomparable PK leading to the successful introduction of new autoinjector devices for monoclonal antibody and Fc-fusion protein products. Information from seven case examples suggests a knowledge gap that warrants attention in autoinjector development.
Antibodies, Monoclonal , Syringes , Humans , Injections, Subcutaneous , Antibodies, Monoclonal/pharmacokinetics , Area Under Curve
OBJECTIVE: Manufacturing changes occur commonly throughout stages of biologics development and may result in product quality attribute changes. As changes in critical quality attributes have the potential to affect clinical safety and efficacy of products, it is imperative to ensure the quality and clinical performance before introducing the after-change products. Thus, we embarked on this project to understand what data have supported the manufacturing changes for licensed products with pre- and post-approval changes. METHODS: We surveyed the manufacturing changes of 85 monoclonal antibodies and 10 Fc fusion proteins approved by the Food and Drug Administration as of December 25, 2021. After collecting the type and timing of changes for these products, we investigated the approaches that provided supporting data for the changes. The source documents included reports submitted by applicants and FDA's regulatory reviews. RESULTS: Analytical comparability was assessed to support all identified manufacturing changes. Supporting clinical data were available in 92% of these manufacturing changes; including data from pharmacokinetic comparability studies alone (3%), other studies on efficacy or safety (70%) and a combination of both (19%). Clinical pharmacokinetic comparability data contributed to supporting substantial changes, such as host cell type or master cell bank changes, concentration or formulation changes, and changes from pre-filled syringes to autoinjectors, especially when introduced after completing pivotal studies. CONCLUSION: Our comprehensive retrospective analysis provides an understanding of the regulatory experience and industry practice, which could facilitate developing appropriate comparability approaches to support manufacturing changes in the future.
Antibodies, Monoclonal , United States , Retrospective Studies , Forecasting , United States Food and Drug Administration
The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th 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 ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 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 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on LBA, Biomarkers/CDx and Cytometry. Part 1 (Mass Spectrometry and ICH M10) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 16 and 14 (2023), respectively.
Biological Assay , Research Report , Flow Cytometry/methods , Ligands , Biomarkers/analysis , Biological Assay/methods
The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th 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 the ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 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 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Mass Spectrometry and ICH M10. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (LBA, Biomarkers/CDx and Cytometry) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 15 and 14 (2023), respectively.
Chromatography , Vaccines , Biomarkers , Cell- and Tissue-Based Therapy , Mass Spectrometry , Oligonucleotides , Technology
US Food and Drug Administration (FDA) guidance outlines how biosimilars can be developed based on pharmacokinetic (PK) and pharmacodynamic (PD) similarity study data in lieu of a comparative clinical efficacy study. There is a paucity of PD comparability studies in biosimilar development, leaving open questions about how best to plan these studies. To that end, we conducted a randomized, double-blinded, placebo-controlled, single-dose, parallel-arm clinical study in healthy participants to evaluate approaches to address information gaps, inform analysis best practices, and apply emerging technologies in biomarker characterization. Seventy-two healthy participants (n = 8 per arm) received either placebo or one of four doses of the proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors alirocumab (15-100 mg) or evolocumab (21-140 mg) to evaluate the maximum change from baseline (ΔPDmax ) and the baseline-adjusted area under the effect curve (AUEC) for the biomarkers low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (apoB) in serum. We investigated approaches to minimize variability in PD measures. Coefficient of variation was lower for LDL-C than apoB at therapeutic doses. Modeling and simulation were used to establish the dose-response relationship and provided support that therapeutic doses for these products are adequately sensitive and are on the steep part of the dose-response curves. Similar dose-response relationships were observed for both biomarkers. ΔPDmax plateaued at lower doses than AUEC. In summary, this study illustrates how pilot study data can be leveraged to inform appropriate dosing and data analyses for a PK and PD similarity study.
Anticholesteremic Agents , Biosimilar Pharmaceuticals , Humans , Biosimilar Pharmaceuticals/adverse effects , PCSK9 Inhibitors , Cholesterol, LDL , Proprotein Convertase 9 , Antibodies, Monoclonal/pharmacokinetics , Pilot Projects , Apolipoproteins B , Biomarkers , Treatment Outcome , Anticholesteremic Agents/pharmacokinetics
The DOSPERT, developed by Weber, Blais and Betz, can be used to measure risk behaviors in a variety of domains. We investigated the use of this scale in China. The participants were 1144 undergraduate students. After we removed some items that were not homogeneous, a principal component analysis extracted six components that accounted for 44.48% of the variance, a value similar to that obtained in the analysis conducted by Weber et al. Chinese undergraduates scored higher on the investment subscale compared with the results of Weber's study. The analysis of individual differences indicated that there was a significant gender difference in the ethical, investment and health/safety subscales, where males scored significantly higher than females. The type of home location was also significant on the ethical and health/safety subscales, where undergraduates from the countryside scored lower than undergraduates from cities and towns on the ethical subscale, and undergraduates from towns scored higher than those from other two areas on the health/safety subscale. Male undergraduates from towns scored higher than male undergraduates from other areas on the gambling subscale.
Risk-Taking , Students/psychology , China , Female , Gambling , Humans , Male , Psychometrics , Sex Factors , Urban Population , Young Adult
The prefusion state of respiratory syncytial virus (RSV) fusion (F) glycoprotein is the target of most RSV-neutralizing activity in human sera, but its metastability has hindered characterization. To overcome this obstacle, we identified prefusion-specific antibodies that were substantially more potent than the prophylactic antibody palivizumab. The cocrystal structure for one of these antibodies, D25, in complex with the F glycoprotein revealed D25 to lock F in its prefusion state by binding to a quaternary epitope at the trimer apex. Electron microscopy showed that two other antibodies, AM22 and 5C4, also bound to the newly identified site of vulnerability, which we named antigenic site Ø. These studies should enable design of improved vaccine antigens and define new targets for passive prevention of RSV-induced disease.
Antibodies, Neutralizing/immunology , Glycoproteins/immunology , Respiratory Syncytial Virus Vaccines/immunology , Respiratory Syncytial Viruses/immunology , Viral Fusion Proteins/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Neutralizing/chemistry , Crystallography, X-Ray , Female , Glycoproteins/chemistry , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Neutralization Tests , Palivizumab , Protein Conformation , Protein Multimerization , Respiratory Syncytial Virus Vaccines/chemistry , Respiratory Syncytial Viruses/physiology , Viral Fusion Proteins/chemistry , Virus Internalization
Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.
Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Evolution, Molecular , HIV Antibodies/chemistry , HIV Antibodies/immunology , HIV-1/chemistry , HIV-1/immunology , AIDS Vaccines/immunology , Africa , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/genetics , CD4 Antigens/chemistry , CD4 Antigens/immunology , Cell Lineage , Cells, Cultured , Clone Cells/cytology , Cross Reactions/immunology , Crystallography, X-Ray , Epitopes/chemistry , Epitopes/immunology , HIV Antibodies/genetics , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp120/metabolism , HIV-1/classification , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Neutralization Tests , Phylogeny , Protein Structure, Tertiary
The SUN (Sad1-UNC-84 homology) domain is conserved in a number of nuclear envelope proteins involved in nuclear migration, meiotic telomere tethering, and antiviral responses. The LINC (linker of nucleoskeleton and cytoskeleton) complex, formed by the SUN and the nesprin proteins at the nuclear envelope, serves as a mechanical linkage across the nuclear envelope. Here we report the crystal structure of the SUN2 protein SUN domain, which reveals a homotrimer. The SUN domain is sufficient to mediate binding to the KASH (Klarsicht, ANC-1, and Syne homology) domain of nesprin 2, and the regions involved in the interaction have been identified. Binding of the SUN domain to the KASH domain is abolished by deletion of a region important for trimerization or by point mutations associated with nuclear migration failure. We propose a model of the LINC complex, where the SUN and the KASH domains form a higher ordered oligomeric network in the nuclear envelope. These findings provide the structural basis for understanding the function and the regulation of the LINC complex.
Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Nuclear Envelope/chemistry , Nuclear Envelope/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Cytoskeleton/metabolism , HEK293 Cells , Humans , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Tertiary
In mitosis, the duplicated chromosomes are separated and equally distributed to progeny cells under the guidance of the spindle, a dynamic microtubule network. Previous studies revealed a mitotic checkpoint that prevents segregation of the chromosomes until all of the chromosomes are properly attached to microtubules through the kinetochores. A variety of kinetochore-localized proteins, including Mad2 and Cdc20, have been implicated in controlling the mitotic checkpoint. Here we report that both Mad2 and Cdc20 can physically associate with Nek2, a serine/threonine kinase implicated in centrosome functions. We show that, similar to Nek2, the endogenous Cdc20 protein can be detected in the centrosome and the spindle poles. Both Cdc20 and Mad2 can be phosphorylated by Nek2. Moreover, our studies demonstrate that overexpression of Nek2 enhances the ability of Mad2 to induce a delay in mitosis. These observations indicate that Nek2 may act upon the Mad2-Cdc20 protein complex and play a critical role in regulating the mitotic checkpoint protein complex. We propose that overexpression of Nek2 may promote aneuploidy by disrupting the control of the mitotic checkpoint.
Aneuploidy , Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/metabolism , Neoplasms/genetics , Repressor Proteins/metabolism , Calcium-Binding Proteins/genetics , Cdc20 Proteins , Cell Cycle , Cell Cycle Proteins/genetics , Cell Division , Cell Line , Gene Duplication , Humans , Mad2 Proteins , Mitosis/physiology , NIMA-Related Kinases , Neoplasms/pathology , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Repressor Proteins/genetics , Sequence Deletion , Spindle Apparatus/genetics
The nuclear envelope forms a selective barrier that separates the cytoplasm from the nucleus. During mitosis the nuclear envelope breaks down so that the microtubule network can form contacts with the kinetochore and guide chromosome segregation. Previous studies have suggested a model in which the centrosome and the microtubule network may play a role in nuclear envelope breakdown through as yet unidentified interactions with proteins localized to the nuclear envelope. In the current study we characterized a nuclear envelope protein SUN2 and identified a substructure involved in its localization to the nuclear envelope. We found that a structurally related protein, SUN1, may be localized to the nuclear envelope through a different mechanism. Furthermore, the SUN2 protein can form different assemblies, including homodimers and heterodimers with SUN1. Finally, we provide evidence indicating that SUN1 and SUN2 may form a physical interaction between the nuclear envelope and the centrosome.
Centrosome/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Nuclear Envelope/metabolism , Animals , Caenorhabditis elegans , Cell Line, Tumor , Cell Nucleus/metabolism , Centrosome/metabolism , Centrosome/ultrastructure , Cloning, Molecular , Cytoplasm/metabolism , HeLa Cells , Humans , Mice , Nuclear Proteins , Protein Binding
DIPA (delta-interacting protein A) was initially identified as a protein that associates with the hepatitis delta antigen. In this study, we found that DIPA can associate with p78/MCRS/MSP58, a Forkhead-associated domain containing protein implicated in malignant transformation as well as in regulation of gene transcription and translation. We analyzed the interaction between DIPA and p78 by co-immunoprecipitation and identified the structural regions involved in the interaction. Consistent with the physical interaction, we found that DIPA is predominant co-localized with p78 to the nucleus. In addition, a fraction of DIPA can be detected on the centrosome. Furthermore, we demonstrate that DIPA can act as a repressor of gene transcription, an activity that appears to be enhanced by p78. Taken together, our results revealed a novel protein complex that plays a role in regulation of gene expression and cell proliferation. We propose that dysfunction of DIPA may contribute to malignant transformation by affecting the functions of p78.
Carrier Proteins/metabolism , Centrosome/metabolism , Nuclear Proteins/metabolism , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , Transcription, Genetic , Adaptor Proteins, Signal Transducing , Down-Regulation , Humans , Immunoprecipitation , Nuclear Proteins/chemistry , Protein Binding , Protein Interaction Mapping , Protein Transport , RNA-Binding Proteins/chemistry , Recombinant Fusion Proteins/metabolism
The centrosome functions as the major microtubule-organizing center and plays a vital role in guiding chromosome segregation during mitosis. Centrosome abnormalities are frequently seen in a variety of cancers, suggesting that dysfunction of this organelle may contribute to malignant transformation. In our efforts to identify the protein components of the centrosome and to understand the structure features involved in the assembly and functions of this organelle, we cloned and characterized a centrosome-associated protein called Su48. We found that a coiled coil-containing subdomain of Su48 was both sufficient and required for its centrosome localization. In addition, this structure also modulates Su48 dimerization. Moreover, ectopic expression of Su48 causes abnormal mitosis, and a mutant form of Su48 disrupts the localization of gamma-tubulin to the centrosome. Finally, by microinjection of an anti-Su48 antibody, we found that disruption of normal Su48 functions leads to mitotic failure, possibly due to centrosome defects or incomplete cytokinesis. Thus, Su48 represents a previously unrecognized centrosome protein that is essential for cell division. We speculate that Su48 abnormalities may cause aberrant chromosome segregation and may contribute to aneuploidy and malignant transformation.
Cell Division/physiology , Centrosome/metabolism , Cytoskeletal Proteins/metabolism , Animals , Cell Line , Cloning, Molecular , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Dimerization , Female , HeLa Cells , Humans , In Vitro Techniques , Male , Mice , Mitosis , Mutagenesis , Pregnancy , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Subcellular Fractions/metabolism , Tissue Distribution , Two-Hybrid System Techniques
Ubiquitously expressed transcript (UXT) is a prefoldinlike protein that has been suggested to be involved in human tumorigenesis. Here, we have found that UXT is overexpressed in a number of human tumor tissues but not in the matching normal tissues. We demonstrate that UXT is located in human centrosomes and is associated with gamma-tubulin. In addition, overexpression of UXT disrupts centrosome structure. Furthermore, abrogation of UXT protein expression by small interfering RNA knockdown leads to cell death. Together, our findings suggest that UXT is a component of centrosome and is essential for cell viability. We propose that UXT may facilitate transformation by corrupting regulated centrosome functions.
Cell Survival/physiology , Centrosome/physiology , Neoplasm Proteins/physiology , Amino Acid Sequence , Bone Neoplasms , Cell Cycle Proteins , Cell Line, Tumor , Centrosome/ultrastructure , Genetic Vectors , Humans , Microscopy, Electron , Molecular Chaperones , Molecular Sequence Data , Mutagenesis , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Osteosarcoma
We have disabled TNF receptor (TNFR) function by inducing allosteric modulation of tryptophan-107 (W107) in the receptor. The allosteric effect operates by means of an allosteric cavity found a short distance from a previously identified loop involved in ligand binding. Occupying this cavity by small molecules leads to perturbation of distal W107 and disables functions of the TNFR, a molecule not known to undergo conformational change upon binding TNF-alpha. TNF-alpha-induced NF-kappaB and p38 kinase activities and clinical symptoms of collagen-induced arthritis in mice were all diminished. Thus, disabling receptor function by induced conformational changes of active binding surfaces represents an innovative paradigm in structure-based drug design.
Receptors, Tumor Necrosis Factor, Type I/chemistry , Receptors, Tumor Necrosis Factor, Type I/metabolism , Allosteric Site/genetics , Amino Acid Substitution , Animals , Arthritis, Experimental/genetics , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Arthritis, Experimental/prevention & control , Base Sequence , Cell Line , DNA/genetics , Humans , In Vitro Techniques , Male , Mice , Mice, Inbred DBA , Models, Molecular , Mutagenesis, Site-Directed , NF-kappa B/metabolism , Protein Conformation , Receptors, Tumor Necrosis Factor, Type I/antagonists & inhibitors , Receptors, Tumor Necrosis Factor, Type I/genetics , Signal Transduction , Tryptophan/chemistry , Tumor Necrosis Factor-alpha/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
The centrosome is a unique organelle that functions as the microtubule organizing center in most animal cells. During cell division, the centrosomes form the poles of the bipolar mitotic spindle. In addition, the centrosomes are also needed for cytokinesis. Each mammalian somatic cell typically contains one centrosome, which is duplicated in coordination with DNA replication. Just like the chromosomes, the centrosome is precisely reproduced once and only once during each cell cycle. However, it remains a mystery how this protein-based structure undergoes accurate duplication in a semiconservative manner. Intriguingly, amplification of the centrosome has been found in numerous forms of cancers. Cells with multiple centrosomes tend to form multipolar spindles, which result in abnormal chromosome segregation during mitosis. It has therefore been postulated that centrosome aberration may compromise the fidelity of cell division and cause chromosome instability. Here we review the current understanding of how the centrosome is assembled and duplicated. We also discuss the possible mechanisms by which centrosome abnormality contributes to the development of malignant phenotype.
Centrosome/physiology , Mitosis/physiology , Models, Biological , Spindle Apparatus/physiology , Aneuploidy , Aurora Kinases , CDC2-CDC28 Kinases/physiology , Cell Cycle Proteins , Cell Division/physiology , Cell Transformation, Neoplastic/pathology , Chromosomal Instability/physiology , Cyclin-Dependent Kinase 2 , DNA Replication/physiology , Protein Serine-Threonine Kinases , Protein Tyrosine Phosphatases , Saccharomyces cerevisiae Proteins
There is an increasing interest in the use of lanthanides in medicine. However, the mechanism of their accumulation in cells is not well understood. Lanthanide cations are similar to ferric ions with regard to transferrin binding, suggesting transferrin-receptor mediated transport is possible; however, this has not yet been confirmed. In order to clarify this mechanism, we investigated the binding of Yb3+ to apotransferrin by UV-Vis spectroscopy and stopped-flow spectrophotometry, and found that Yb3+ binds to apotransferrin at the specific iron sites in the presence of bicarbonate. The apparent binding constants of these sites showed that the affinity of Yb3+ is lower than that of Fe3+and binding of Yb3+ in the N-lobe is kinetically favored while the C-lobe is thermodynamically favored. The first Yb3+ bound to the C-lobe quantitatively with a Yb/apotransferrin molar ratio of < 1, whereas the binding to the other site is weaker and approaches completeness by a higher molar ratio only. As demonstrated by 1H NMR spectra, Yb3+ binding disturbed the conformation of apotransferrin in a manner similar to Fe3+. Flow cytometric studies on the uptake of fluorescein isothiocyanate labeled Yb3+-bound transferrin species by K562 cells showed that they bind to the cell receptors. Laser scanning confocal microscopic studies with fluorescein isothiocyanate labeled Yb3+-bound transferrin and propidium iodide labeled DNA and RNA in cells indicated that the Yb3+ entered the cells. The Yb3+-transferrin complex inhibited the uptake of the fluorescein labeled ferric-saturated transferrin (Fe2-transferrin) complex into K562 cells. The results demonstrate that the complex of Yb3+-transferrin complex was recognized by the transferrin receptor and that the transferrin-receptor-mediated mechanism is a possible pathway for Yb3+ accumulation in cells.
Transferrin/metabolism , Ytterbium/chemistry , Binding, Competitive , Cations , Cell Line , Cell Membrane/metabolism , Flow Cytometry , Humans , Iron , K562 Cells , Kinetics , Magnetic Resonance Spectroscopy , Microscopy, Confocal , Protein Binding , Protein Conformation , Spectrophotometry , Thermodynamics , Transferrin/chemistry , Ultraviolet Rays , Ytterbium/metabolism
