Assuntos
Neoplasias/genética , Publicações Periódicas como Assunto/tendências , Publicações/estatística & dados numéricos , Editoração/tendências , Pesquisadores/normas , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Políticas Editoriais , Humanos , Internet , Neoplasias/diagnóstico , Neoplasias/terapia , Publicações Periódicas como Assunto/normas , Publicações/normas , Editoração/normas , Ciência/normasRESUMO
PURPOSE: To study the kinetics of gene expression alterations following radiation exposure of isogenic HCT116 p53 +/+ and HCT116 p53-/- cell lines. MATERIALS AND METHODS: Cells were exposed to 5 Gy of irradiation (Cs-137) and genome-wide temporal expression analysis using Illumina bead chip arrays was performed. Signalling pathways were explored using Metacore (Genego). Biological responses including cell cycle checkpoint activation, centrosome amplification and senescence induction were analyzed. RESULTS: Significant differences in the radiation response were observed between the p53+/+ and the p53-/- cell lines. In p53+/+ cells concurrent G1- and G2-arrests were activated followed by senescence induction. Increased expression of genes associated with senescence, senescence associated secretory phenotype (SASP) and repression of genes essential for G2-M transition were detected. P53-/- cells arrested mainly in G2 followed by centrosome amplification, mitotic slippage and a subsequent increase of polyploid cells. Furthermore, changes in expression correlated well with these signs of mitotic catastrophe. CONCLUSIONS: The presence or absence of p53 triggers different signalling cascades with different endpoints. Elucidating these differences is important as it enables improvement of radiation treatment and could be used to develop new combination treatments with specific inhibitors of key regulators of these cell death modalities.
Assuntos
Neoplasias Colorretais/patologia , Dano ao DNA , Perfilação da Expressão Gênica , Genômica , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Senescência Celular/efeitos da radiação , Centrossomo/efeitos da radiação , Relação Dose-Resposta à Radiação , Células HCT116 , Humanos , Cinética , Mitose/efeitos da radiação , FenótipoRESUMO
AIM: To explore kinetic changes in the gene expression profile during radiation-induced mitotic catastrophes. MATERIALS AND METHODS: Gene expression changes were measured in HPV-infected HeLa Hep2 tumor cells following exposure to 5 Gy of ionizing radiation ((60)Co). Signaling pathways were explored and correlated to the biological responses linked to mitotic catastrophe. RESULTS: Following irradiation a transient G2-arrest was induced. Anaphase bridge formation and centrosome hyperamplification was observed. These phenotypical changes correlated well with the observed gene expression changes. Genes with altered expression were found to be involved in mitotic processes as well as G2- and spindle assembly checkpoints. Also centrosome-associated genes displayed an increased expression. CONCLUSION: This study elucidates specific characteristics in the altered gene expression pattern induced by irradiation, which can be correlated to the events of mitotic catastrophe in HeLa Hep2 cells. Therapeutic strategies modulating these alterations might potentiate future therapy and enhance tumor cell killing.
Assuntos
Mitose/efeitos da radiação , Transcriptoma/efeitos da radiação , Centrossomo/efeitos da radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Regulação Neoplásica da Expressão Gênica , Células HeLa , HumanosRESUMO
This study aims to identify the temporal changes in gene expression in MOLT-4, a leukemia cell line, in response to radiation and to present a comprehensive description of the pathways and processes that most significantly relate to the cellular biological responses. A global gene expression profile of 24,500 genes was performed on MOLT-4 tumor cells following exposure to 5 Gy of ionizing radiation ((60)Co) using a bead chip array (Illumina). Signaling pathways and processes significantly altered following irradiation were explored using MetaCore. Cellular viability [3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], activation of cell cycle checkpoints [fluorescence activated cell sorting (FACS)], and induction of apoptosis (FACS, caspase assays) were evaluated to correlate these biological responses to the gene expression changes. Totally, 698 different genes displayed a significantly altered expression following radiation, and out of these transcripts, all but one showed increased expression. One hour following irradiation, the expression was changed only for a few genes. Striking changes appeared at later time-points. From 3 to 24 h post-irradiation, a significant fraction of the genes with altered expression were found to be involved in cell cycle checkpoints and their regulation (CDKN1A), DNA repair (GADD45A, DDB2, XPC), apoptosis induction (DR5, FasR, Apo-2L, Bax), and T-cell activation/proliferation (CD70, OX40L). Irradiated MOLT-4 cells were arrested at the G2-checkpoint, followed by a decrease in cell viability, most pronounced 48 h after exposure. The cell death was executed by induced apoptosis and was visualized by an increase in subG1 cells and an increased activation of initiator (caspase-8 and caspase-9) and execution (caspase-3) caspases. Activation of cell cycle arrest and apoptosis correlated well in time with the changes in gene expression of those genes important for these biological processes. Activation of the apoptotic signaling pathways in MOLT-4 cells following irradiation includes components from the intrinsic as well as the extrinsic apoptotic pathways. This study indicates that the altered gene expression pattern induced by irradiation is important for the sequential steps observed in MOLT-4 cells during apoptosis induction.
Assuntos
Apoptose/efeitos da radiação , Raios gama/efeitos adversos , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos da radiação , Leucemia de Células T/genética , Ciclo Celular/efeitos da radiação , Morte Celular/efeitos da radiação , Linhagem Celular Tumoral , Humanos , Transdução de Sinais/efeitos da radiação , Fatores de TempoRESUMO
Fourteen monoclonal antibodies with specificity against native or recombinant antigens within the S100 family were investigated with regard to immunoreactivity. The specificities of the antibodies were studied using ELISA tests, Western blotting epitope mapping using competitive assays, and QCM technology. The mimotopes of antibodies against S100A4 were determined by random peptide phage display libraries. Antibody specificity was also tested by IHC and pair combinations evaluated for construction of immunoradiometric assays for S100B. Out of the 14 antibodies included in this report eight demonstrated specificity to S100B, namely MAbs 4E3, 4D2, S23, S53, 6G1, S21, S36, and 8B10. This reactivity could be classified into four different epitope groups using competing studies. Several of these MAbs did display minor reactivity to other S100 proteins when they were presented in denatured form. Only one of the antibodies, MAb 3B10, displayed preferential reactivity to S100A1; however, it also showed partial cross-reactivity with S100A10 and S100A13. Three antibodies, MAbs 20.1, 22.3, and S195, were specific for recombinant S100A4 in solution. Western blot revealed that MAb 20.1 and 22.3 recognized linear epitopes of S100A4, while MAb S195 reacted with a conformational dependent epitope. Surprisingly, MAb 14B3 did not demonstrate any reactivity to the panel of antigens used in this study.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas Recombinantes/imunologia , Proteínas S100/imunologia , Animais , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Western Blotting , Bovinos , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Humanos , Imunoensaio , Técnicas Imunoenzimáticas , Biblioteca de Peptídeos , Radioimunoensaio , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas S100/química , Proteínas S100/metabolismoRESUMO
Recombinant antibodies may be engineered to obtain improved functional properties. Functional mapping of the residues in the binding surfaces is of importance for predicting alterations needed to yield the desired properties. In this investigation, 17 single mutation mutant single-chain variable fragments (scFvs) of the anti-idiotypic antibody anti-TS1 were generated in order to functionally map amino acid residues important for the interaction with its idiotype TS1. Residues in anti-TS1 determined to be very important for the interaction were identified, Y32L, K50L, K33H, and Y52H, and they were distributed adjacent to a centrally located hydrophobic area, and contributed extensively to the interaction energy (≥2.5 kcal/mol) in the interaction. Quantitative ELISA assays, BIAcore technologies and three-dimensional surface analysis by modeling were employed to visualize the consequences of the mutations. The expression levels varied between 2 - 1,800 nM as determined by ELISA. All the 17 scFvs displayed higher dissociation rates (60 - 1,300 times) and all but two of them also faster association rates (1.3 - 56 times). The decrease in affinity was determined to be 1.6 - 12,200 times. Two of the mutants displayed almost identical affinity with the wild type anti-TS1, but with a change in both association and dissociation rates. The present investigation demonstrates that it is possible to generate a large panorama of anti-idiotypic antibodies, and single out a few that might be of potential use for future clearing and pre-targeting purposes of idiotypic-anti-idiotypic interactions.
Assuntos
Anticorpos Anti-Idiotípicos/química , Região Variável de Imunoglobulina/química , Anticorpos de Cadeia Única/química , Sequência de Aminoácidos , Anticorpos Anti-Idiotípicos/imunologia , Afinidade de Anticorpos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Humanos , Hibridomas , Região Variável de Imunoglobulina/imunologia , Queratina-8/imunologia , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Quaternária de Proteína , Anticorpos de Cadeia Única/imunologiaRESUMO
Recombinant single-chain fragment variable (scFv) antibodies with specificity to tumor antigens can be used to target tumors in vivo. The approach to use administration of complexes of idiotypic-anti-idiotypic scFvs when targeting tumors has not been tested earlier, and from a theoretical point it could contribute to longer in vivo circulation and improved targeting efficiency by dissociation, when in contact with the target antigen. In this study two models to evaluate the targeting efficiency of such complexes were used. HeLa HEp-2 tumor cells were grown as multicellular tumor spheroids (MCTS) and exposed to the antibody constructs in vitro. The behavior in vivo was tested in an in vivo tumor xenograft model. To increase the size of the anticytokeratin 8 scFv, TS1-218, complexes were formed between TS1-218 and its anti-idiotype, alphaTS1 scFv. The functionality of (125)I-labeled TS1-218 alone and in complex was studied in both models. The uptake patterns were similar in both models. The idiotypic TS1-218 was able to localize to the MCTS and xenografted tumors, both alone and in complex with alphaTS1 scFv. TS1-218 in complex, however, demonstrated a significantly higher uptake than the monomeric TS1-218 in both models (p < 0.0005 and p < 0.0089, respectively). When complexes were administered in vivo, a slower clearance and an increased tumor half-life could be observed. The present investigation indicates that administration of targeting antibodies, with initially blocked antigen-binding sites by complex formation with their anti-idiotypes, may improve targeting efficiency.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/farmacocinética , Radioisótopos do Iodo/farmacocinética , Queratina-8/imunologia , Neoplasias Experimentais/diagnóstico por imagem , Anticorpos de Cadeia Única/imunologia , Esferoides Celulares/diagnóstico por imagem , Animais , Feminino , Células HeLa , Humanos , Idiótipos de Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Cintilografia , Distribuição TecidualRESUMO
The main goal when treating malignancies with radiation therapy is to deprive tumor cells of their reproductive potential. One approach to achieve this is by inducing tumor cell apoptosis. Accumulating evidences suggest that induction of apoptosis alone is insufficient to account for the therapeutic effect of radiotherapy. It has become obvious in the last few years that inhibition of the proliferative capacity of malignant cells following irradiation, especially with solid tumors, can occur via alternative cell death modalities or permanent cell cycle arrests, i.e., senescence. In this review, apoptosis and mitotic catastrophe, the two major cell deaths induced by radiation, are described and dissected in terms of activating mechanisms. Furthermore, treatment-induced senescence and its relevance for the outcome of radiotherapy of cancer will be discussed. The importance of p53 for the induction and execution of these different types of cell deaths is highlighted. The efficiency of radiotherapy and radioimmunotherapy has much to gain by understanding the cell death mechanisms that are induced in tumor cells following irradiation. Strategies to use specific inhibitors that will manipulate key molecules in these pathways in combination with radiation might potentiate therapy and enhance tumor cell kill.
Assuntos
Apoptose/efeitos da radiação , Mitose/efeitos da radiação , Neoplasias/radioterapia , Animais , Senescência Celular/efeitos da radiação , Humanos , Proteína Supressora de Tumor p53/fisiologiaRESUMO
BACKGROUND: The apoptotic signalling pathways involved in the delayed type of apoptosis occurring in HeLa Hep2 cells following radiation were investigated. MATERIALS AND METHODS: HeLa Hep2 cells were exposed to 5 Gy of cobalt-60 radiation. The activation of caspase-2, caspase-8, caspase-9 and effector caspase-3 was investigated by caspase assay plates and Western blots. Cleavage of poly (ADP-ribose) polymerase (PARP) was analysed on Western blots. HeLa Hep2 cells were irradiated with or without preincubation with inhibitors of protein synthesis (cycloheximide, CHX) and caspases, followed by TUNEL staining and caspase assay plate evaluation. RESULTS: Initiator caspases-2, -8, -9, and effector caspase-3, were found to be activated and PARP cleaved following irradiation. CHX completely inhibited the caspase activation and the associated apoptosis. Pretreatment with caspase-2 inhibitor indicated that caspase-2 was involved in the execution of the apoptosis. CONCLUSION: Activation of the apoptotic signalling pathways following irradiation of HeLa Hep2 cells includes components from the intrinsic as well as the extrinsic pathways and seems to require de novo protein synthesis.
Assuntos
Adenocarcinoma/patologia , Apoptose/efeitos da radiação , Neoplasias do Colo do Útero/patologia , Adenocarcinoma/enzimologia , Adenocarcinoma/radioterapia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caspase 2/metabolismo , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular Tumoral , Radioisótopos de Cobalto , Ativação Enzimática/efeitos da radiação , Feminino , Raios gama , Células HeLa , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais/efeitos da radiação , Neoplasias do Colo do Útero/enzimologia , Neoplasias do Colo do Útero/radioterapiaRESUMO
Iodine-131 (131I) has been used both in unconjugated form and conjugated to antibody derivates (i.e., radioimmunotherapy; RIT) to treat malignant diseases. The mechanisms by which 131I-irradiation causes growth retardation are, however, inadequately understood. The aim of this study was to elucidate the sequential molecular and cellular events that initiate cell death in HeLa Hep2 cells exposed to 131I. In this paper, HeLa Hep2 cells were found to display a transient G2-M arrest following irradiation, but then reentered the cell cycle still containing unrepaired cellular damage. An increase of multipolar mitotic spindles, as well as a significant increase in centrosome numbers from 8.8% +/- 1.9% in controls to 54.7% +/- 2.2% in irradiated cells, was observed (p < 0.0001). A subsequent failure of cytokinesis caused the cells to progress into mitotic catastrophe. This was accompanied by the formation of giant cells with multiple nuclei, multilobulated nuclei, and an increased frequency of polyploidy cells. A fraction of the cells also displayed apoptotic features, including the activation of initiator caspases-2, -8, -9, and effector caspase-3, as well as cleavage of poly(ADP-ribose) polymerase, a cell-death substrate for active caspase-3. These findings demonstrate that mitotic catastrophes and the activation of a delayed type of apoptosis might be important mechanisms involved in cell death following the RIT of solid tumors with -emitting radionuclides, such as 131I.
Assuntos
Apoptose , Radioisótopos do Iodo/farmacologia , Mitose , Neoplasias/radioterapia , Caspases/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Centrossomo/efeitos da radiação , Ativação Enzimática , Citometria de Fluxo/métodos , Células HeLa , Humanos , Microscopia de Fluorescência/métodos , Poli(ADP-Ribose) Polimerases/metabolismo , Radioimunoterapia/métodosRESUMO
PURPOSE: Experimental radioimmunotherapy delivering absorbed doses of 2.5 to 10 Gy has been shown to cause growth retardation of tumors. The purpose of this study was to elucidate the sequential molecular and cellular events occurring in HeLa Hep2 cells exposed to such doses. METHODS: Dose-response curves, activation of cell cycle checkpoints, and mitotic behavior were investigated in HeLa Hep2 cells following 2.5- to 10-Gy irradiation by carrying out 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, Western blots, fluorescence-activated cell sorting analysis, and immunofluorescence stainings. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining was used to detect apoptosis. RESULTS: A G2-M arrest was shown by fluorescence-activated cell sorting analysis. p53 and p21 were found to be up-regulated but were not immediately related to the arrest. The G2-M arrest was transient and the cells reentered the cell cycle still containing unrepaired cellular damage. This premature entry caused an increase of anaphase bridges, lagging chromosomal material, and multipolar mitotic spindles as visualized by propidium iodide staining and immunofluorescence staining with alpha-tubulin and gamma-tubulin antibodies. Furthermore, a dose-dependent significant increase in centrosome numbers from 12.6+/-6.6% to 67+/-5.3% was identified as well as a dose-dependent increase of polyploid cells from 2.8+/-1.3% to 17.6+/-2.1% with the highest absorbed dose of 10 Gy. These disturbances caused the cells to progress into mitotic catastrophe and a fraction of these dying cells showed apoptotic features as displayed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining 5 to 7 days after irradiation. CONCLUSION: An absorbed dose of 2.5 to 10 Gy was shown to force HeLa Hep2 cells into mitotic catastrophe and delayed apoptosis. These might be important cell death mechanisms involved in tumor growth retardation following radioimmunotherapy of solid tumors.
Assuntos
Divisão Celular/efeitos da radiação , Fase G2/efeitos da radiação , Raios gama , Mitose/efeitos da radiação , Apoptose/efeitos da radiação , Western Blotting , Divisão Celular/fisiologia , Radioisótopos de Cobalto , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Relação Dose-Resposta à Radiação , Citometria de Fluxo , Fase G2/fisiologia , Células HeLa/efeitos da radiação , Humanos , Marcação In Situ das Extremidades Cortadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
One single-chain Fv antibody fragment (scFv) and a new recombinant covalently linked dimeric scFv antibody (sc(Fv)(2)) against placental alkaline phosphatase (PLAP) were investigated for selective tumor targeting. The biological behavior of these new antibodies was compared to that of the original native antibody, H7 MAb. The sc(Fv)(2)) antibody displayed convincing tumor localization properties with a rapid excretion pattern comparable to the scFv, but with a longer retention time in the tumor, and higher tumor-to-nontumor ratio (27:1), compared to the scFv (15:1), at 48 hours. For the sc(Fv)(2) antibody, more than 50% of the remaining activity in the mouse was present in the tumor between 24 and 48 hours after the injection. With this antibody, scintigraphic visualization of the tumor was also possible 1 week after the injection. It is concluded that this sc(Fv)(2) antibody fragment, with two binding sites, displays properties suitable for in vivo targeting of PLAP expressing tumors.
Assuntos
Fosfatase Alcalina/imunologia , Anticorpos Monoclonais/imunologia , Neoplasias/diagnóstico por imagem , Neoplasias/imunologia , Placenta/imunologia , Radioimunodetecção/métodos , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Frontotemporal dementia (FTD) is pathologically heterogeneous, sometimes revealing intraneuronal inclusions of neurofilaments. We therefore measured CSF neurofilament profiles in patients with FTD, patients with early onset Alzheimer's disease (EAD) and healthy control subjects to explore the discriminative potential of CSF neurofilaments compared with the existing CSF biomarkers amyloid-beta(1-42), tau and tau phosphorylated at threonine-181. METHODS: CSF levels of light chain, heavy chain and hyperphosphorylated heavy chain neurofilaments (NfL, t-NfH and P-NfH) were compared between 17 subjects with FTD, 20 with EAD and 25 cognitively healthy controls. RESULTS: A subgroup of FTD patients had remarkably high CSF levels of both NfL and NfH. The degree of NfH phosphorylation was increased in FTD compared to both other groups. The levels of CSF NfL were significantly higher in EAD compared to controls. CONCLUSION: Differences in CSF biomarker profiles might reflect differential involvement of neurofilaments and tau in FTD and EAD. The subgroup of FTD patients with high CSF neurofilament levels may have a different neuropathological substrate and future studies addressing this specific issue are needed.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Demência/líquido cefalorraquidiano , Proteínas de Neurofilamentos/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Idoso , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Peptídeos beta-Amiloides/metabolismo , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/metabolismo , Demência/patologia , Feminino , Humanos , Masculino , Análise por Pareamento , Pessoa de Meia-Idade , Proteínas de Neurofilamentos/metabolismo , Fragmentos de Peptídeos/líquido cefalorraquidiano , Fragmentos de Peptídeos/metabolismo , Fosforilação , Valores de Referência , Proteínas tau/metabolismoRESUMO
A new, easier and efficient purification method, using Sephacryl and DEAE-Sephacel, of the C-terminal fragment of two alpha-macroglobulins, alpha(2)-M and PZP, is presented. Two larger peptides were identified for each protein as the C-terminal fragment, with molecular weights of approximately 30 kDa and the N-terminal sequences were determined to be SSTQDTV for alpha(2)-M and VALHLS for PZP. The smaller peptides with molecular weights of 18 kDa correspond to a shorter C-terminal sequence of these proteins, and they were determined to be EEFPFA for alpha(2)-M and ALKVQTV for PZP, with no interfering sequences detected. The results confirmed the discriminatory capacity of the purification procedure and the purity of the fragments. This new methodology facilitates biological studies of alpha-macroglobulins, and will enable elucidation of the role the C-terminal region may exert to eliminate alpha-macroglobulin-proteinases complexes from the circulation by the LRP/receptor.
Assuntos
Endopeptidases/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas da Gravidez/química , alfa-Macroglobulinas/química , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Western Blotting , Quimotripsina/farmacologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hidrólise , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Gravidez , Proteínas da Gravidez/metabolismo , Estrutura Terciária de Proteína , Análise de Sequência de Proteína , Fatores de Tempo , alfa-Macroglobulinas/metabolismoRESUMO
Multivalency is a recognized means to increase the functional affinity of single-chain antibody fragments (scFvs) for optimizing tumor uptake at radioimmunotargeting. A unique divalent tandem single-chain Fv antibody [sc(Fv)2], based on the variable regions of the monoclonal antibody (MAb) H7, has now been generated. The antibody differs from other dimeric single-chain constructs in that a linker sequence (L) is introduced between the repeats of VL and VH domains (VL-L-VH-L-VL-L-VH). This construct was expressed as a His-tagged TrxA fusion protein in the Escherichia coli strain Origami B. Following cleavage with AcTev protease, the antibody was obtained in soluble and active form in E. coli and could be purified by Ni-NTA and cation-exchange chromatography. Purity and immunochemical properties were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), Western blot, and Biacore analyses. The [sc(Fv)2] displayed proper stability and could be purified to homogeneity. This antibody also maintained immunoreactivity at 42 degrees C with only slight decrease at 52 degrees C. The high affinity displayed by the original antibody H7, 6.7 x 10(9) M(-1), was only slightly decreased to 1.2 x 10(9) M(-1) as determined by Biacore. The generation of such a divalent single-chain Fv with a molecular weight around 60 kd would be of value for clinical applications such as radioimmunolocalization and radioimmunotherapy.
Assuntos
Anticorpos/genética , Anticorpos/isolamento & purificação , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Isoenzimas/imunologia , Proteínas Recombinantes de Fusão/síntese química , Fosfatase Alcalina , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli , Proteínas Ligadas por GPI , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/isolamento & purificação , Sequências de Repetição em Tandem/genéticaRESUMO
Recombinant technologies to engineer ordinary hybridoma monoclonal antibodies (MAbs) to single-chain fragment variable (scFv) may cause loss of antibody affinity, increased tendency to aggregate, increased temperature sensitivity, and low yield of active protein. In the present investigation, the well-characterized MAb H7 against placental alkaline phosphatase (PLAP), used as a model antibody, was engineered to improve solubility and stability of scFv with retained high affinity. The original procedure to generate single-chain antibodies with a 10-amino acid linker between VH and VL yielded an almost insoluble product. By site-directed mutagenesis, four selective sequence substitutions were made in the VL fragment and one in the VH fragment to improve solubility. The importance of the linker length was investigated, and a 25/30 amino acid linker was found to improve solubility. In order to further increase the stability of the single-chain antibody, an additional covalent -S-S- bond was introduced between amino acid 100 in the VL fragment and amino acid 44 in the VH region, to make a single-chain disulphide stabilized variable fragment (scdsFv). Altogether five different antibody constructs were produced and compared in terms of solubility, stability, affinity, and production properties. Immunospecificity was tested by enzyme-linked immunosorbent assay (ELISA) against the target antigen, temperature sensitivity by exposing the purified scFv to higher temperatures. All the new constructs retained almost equal activity and high affinity for their target antigen, placental alkaline phosphatase (PLAP), compared to the intact MAb H7, up to +42 degrees C as evaluated by ELISA. The overall affinity K(A) > 10(9) (M(1)) of the new antibodies could be maintained in the same order of magnitude as the original one (H7), when evaluated by Biacore technology. The best final single-chain antibody was obtained by performing the specific site-directed mutations and introducing a linker of 30 amino acids, but not by additional stabilizing disulphide bonds. The yield of the final antibody was improved approximately 10-fold by the modifications. This antibody could easily be expressed in a bacterial system using the PET-32a TrxA vector and the Escherichia coli strain BL21 Origami B (DE3). Purified antibody, which could be kept at concentrations up to 0.8 mg/mL, was obtained, which is sufficient for clinical testing of therapeutic applications.
Assuntos
Fosfatase Alcalina/imunologia , Anticorpos/genética , Região Variável de Imunoglobulina/genética , Placenta/enzimologia , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Animais , Feminino , Humanos , Hibridomas , Camundongos , Dados de Sequência Molecular , GravidezRESUMO
Strontium is used in the treatment of osteoporosis as a ranelate compound, and in the treatment of painful scattered bone metastases as isotope. At very high doses and in certain conditions, it can lead to osteomalacia characterized by impairment of bone mineralization. The osteomalacia symptoms resemble those of hypophosphatasia, a rare inherited disorder associated with mutations in the gene encoding for tissue-nonspecific alkaline phosphatase (TNAP). Human alkaline phosphatases have four metal binding sites--two for zinc, one for magnesium, and one for calcium ion--that can be substituted by strontium. Here we present the crystal structure of strontium-substituted human placental alkaline phosphatase (PLAP), a related isozyme of TNAP, in which such replacement can have important physiological implications. The structure shows that strontium substitutes the calcium ion with concomitant modification of the metal coordination. The use of the flexible and polarizable force-field TCPEp (topological and classical polarization effects for proteins) predicts that calcium or strontium has similar interaction energies at the calcium-binding site of PLAP. Since calcium helps stabilize a large area that includes loops 210-228 and 250-297, its substitution by strontium could affect the stability of this region. Energy calculations suggest that only at high doses of strontium, comparable to those found for calcium, can strontium substitute for calcium. Since osteomalacia is observed after ingestion of high doses of strontium, alkaline phosphatase is likely to be one of the targets of strontium, and thus this enzyme might be involved in this disease.
Assuntos
Fosfatase Alcalina/química , Calcificação Fisiológica , Osteomalacia/etiologia , Estrôncio/química , Cálcio , Proteínas Ligadas por GPI , Humanos , Isoenzimas , Conformação ProteicaRESUMO
At immunolocalization of experimental tumors, idiotypic monoclonal antibodies, such as TS1 against cytokeratin 8, can be used to carry and deposit in vivo terapeutics in the tumor. These carriers also remain in the circulation and may cause negative side-effects in other tissues. In this report, several derivatives of the antiidiotypic antibody alphaTS1 were produced and tested for their clearing capacity of the idiotypic carrier antibody TS1. Intact monoclonal alphaTS1, scFv of a alphaTS1 and alphaTS1 Fab'2 and fragments were produced by recombinant technology or by cleavage with Ficin. The scFv was tailored by use of the variable domain genes of the light and heavy chain from the hybridoma clone in combination with a (Gly4Ser)3-linker, followed by expression in E. coli. When tested for clearing capacity, the intact divalent antiidiotypic IgG was found to be the most efficient. The divalent and the monovalent Fab fragment also demonstrated significant clearing, but lower than the intact antiidiotypic IgG. The alphaTS1 scFv antibody when injected separately was not found to clear the idiotype, but could do so when preincubated with the idiotype. Rapid excretion and in vivo instability of this low molecular weight antibody fragment may be the major reasons. Similar results were obtained when the system was reversed and the 131I-labeled antiidiotype IgG was cleared with the idiotype fragment. It is concluded that both intact antiidiotypic IgG, and Fab'2 fragments are able to clear the idiotypic antibodies. The experimental data support the conclusion that the Fc parts from both the idiotype and the antiidiotype may contribute to this elimination.