28 results on '"Guikema JEJ"'
Search Results
2. CD27-triggering on primary plasma cell leukaemia cells has anti-apoptotic effects involving mitogen activated protein kinases
- Author
-
Guikema, JEJ, Vellenga, E, Abdulahad, WH, Hovenga, S, Bos, NA, Other departments, Guided Treatment in Optimal Selected Cancer Patients (GUTS), Stem Cell Aging Leukemia and Lymphoma (SALL), and Translational Immunology Groningen (TRIGR)
- Subjects
TERMINAL KINASE ,NF-KAPPA-B ,apoptosis ,p38 ,extracellular-regulated kinase 1/2 ,CD27 EXPRESSION ,MULTIPLE-MYELOMA CELLS ,P38 MAP KINASE ,CD27/CD70 INTERACTION ,COMPARATIVE GENOMIC HYBRIDIZATION ,SIGNAL-REGULATED KINASE ,INDUCED INHIBITION ,plasma cell leukaemia ,GLUCOCORTICOID-INDUCED APOPTOSIS ,CD27 - Abstract
Primary plasma cell leukaemia (PCL) is a rare plasma cell malignancy, which is related to multiple myeloma (MM) and is characterized by a poor prognosis. In a previous study we demonstrated that PCL plasma cells display a high expression of CD27, in contrast to MM plasma cells. The present study was set out to assess the functional properties of CD27 expressed on PCL plasma cells by triggering with its ligand CD70. Using CD27-expressing purified plasma cells from a PCL patient we demonstrated that CD27-triggering modestly inhibited spontaneous and dexamethasone-induced apoptosis. In vitro stimulation and Western blotting showed that activation of p38 and extracellular-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinases (MAPK) was associated with CD27-mediated signal transduction. Specific inhibition of p38 and ERK1/2 MAPK abolished the anti-apoptotic effects of CD27-triggering. Interestingly, simultaneous inhibition of p38 and ERK1/2 strongly sensitized PCL cells for dexamethasone-induced apoptosis. Finally, in dexamethasone-treated PCL cells, CD27-triggering was associated with persistent DNA-binding activity of activator protein 1 (AP-1) but not of nuclear factor-kappaB. These findings suggest that, in primary PCL, specific anti-apoptotic pathways exist that might provide novel therapeutic targets.
- Published
- 2004
3. Heterogeneity of clonal B cells in Multiple Myeloma
- Author
-
Guikema, JEJ, Vellenga, Edo, Nieuwenhuis, P, Bos, Nico, and Faculteit Medische Wetenschappen/UMCG
- Subjects
ComputingMethodologies_DOCUMENTANDTEXTPROCESSING ,GeneralLiterature_REFERENCE(e.g.,dictionaries,encyclopedias,glossaries) - Published
- 2004
4. CD27 is heterogeneously expressed in multiple myeloma: low CD27 expression in patients with high-risk disease
- Author
-
Guikema, JEJ, Hovenga, S, Vellenga, E, Conradie, JJ, Abdulahad, WH, Bekkema, R, Smit, JW, Zhan, FH, Shaughnessy, J, Bos, NA, Other departments, Faculteit Medische Wetenschappen/UMCG, Guided Treatment in Optimal Selected Cancer Patients (GUTS), Stem Cell Aging Leukemia and Lymphoma (SALL), and Translational Immunology Groningen (TRIGR)
- Subjects
cDNA microarray ,ANTIGEN ,chemical and pharmacologic phenomena ,hemic and immune systems ,GENE FAMILY ,multiple myeloma ,DIFFERENTIATION ,immunophenotyping ,immune system diseases ,CD27/CD70 INTERACTION ,MARKER ,MEMORY B-CELLS ,NORMAL PLASMA-CELLS ,GROWTH ,LIGAND ,CD27 ,TUMOR-NECROSIS-FACTOR - Abstract
Expression of CD27 on malignant plasma cells (PC) (CD138(+) CD38(++) ) was analysed in a cross-sectional study of bone marrow (BM) samples from multiple myeloma (MM) patients (n = 28), monoclonal gammopathy of undetermined significance (MGUS) patients (n = 6) and BM PC from healthy donors (n = 4). MM PC expressed CD27 with a variable, lower intensity pattern compared with the consistent high expression in MGUS and healthy donors. MM patients in complete clinical remission displayed a higher percentage of CD27(+) PC compared with patients at diagnosis, relapse or in partial remission. In MM, loss of CD27 correlated with loss of CD19 (R (2) = 0.4, P
- Published
- 2003
5. Myeloma clonotypic B cells are hampered in their ability to undergo B-cell differentiation in vitro
- Author
-
Guikema, JEJ, Vellenga, E, Bakkus, MHC, Bos, NA, Guided Treatment in Optimal Selected Cancer Patients (GUTS), Stem Cell Aging Leukemia and Lymphoma (SALL), and Translational Immunology Groningen (TRIGR)
- Subjects
POLYMERASE-CHAIN-REACTION ,TRANSPLANTATION ,BONE-MARROW ,PROLIFERATION ,differentiation ,PERIPHERAL-BLOOD ,INTERLEUKIN-10 ,multiple myeloma ,MALIGNANT PLASMA-CELL ,MULTIPLE-MYELOMA ,IL-10 ,CD40 ,clonotypic B cell ,SEQUENCES REVEAL - Abstract
In the peripheral blood (PB) of multiple myeloma (MM) patients, clonotypic B cells are present that express the identical V( D) J rearrangements as the malignant plasma cells in the bone marrow. In the present study, the proliferative capacity of clonotypic B cells from MM patients (n = 10) and the ability to differentiate in vitro was determined using the CD40-culturing system. For six patients, the presence of clonotypic B cells expressing variant immunoglobulin (Ig) isotypes was assessed by Ig isotype-specific allele-specific oligonucleotide reverse transcription polymerase chain reaction (ASO-RT-PCR) after culturing with CD40L and interleukin 4 (IL-4). In three out of six patients, clonotypic B cells expressing variant isotypes were detected both before and after culturing. The ability of clonotypic B cells to undergo B-cell differentiation was studied by abrogating CD40 signalling accompanied by IL-10 and IL-2 stimulation, enhancing differentiation towards Ig-secreting cells. The numbers of clonotypic B cells were determined by quantitative ASO-PCR. An increase in cell number was observed upon CD40L and IL-4 stimulation, whereas the relative number of clonotypic B cells was unaltered. In contrast, upon B-cell differentiation the relative number of clonotypic B cells decreased. In conclusion, clonotypic B cells can be cultured and isolated in vitro using the CD40 system. Clonotypic B cells responded to CD40 triggering in a similar fashion as to non-clonotypic normal B cells. However, the ability of clonotypic B cells to undergo in vitro activation and differentiation into Ig-secreting cells is hampered.
- Published
- 2002
6. Multiple myeloma related cells in patients undergoing autologous peripheral blood stem cell transplantation
- Author
-
Guikema, JEJ, Vellenga, E, Veeneman, JM, Hovenga, S, Bakkus, MHC, Klip, H, Bos, NA, Other departments, Faculteit Medische Wetenschappen/UMCG, Cell Biochemistry, Guided Treatment in Optimal Selected Cancer Patients (GUTS), Stem Cell Aging Leukemia and Lymphoma (SALL), and Translational Immunology Groningen (TRIGR)
- Subjects
multiple myeloma ,MALIGNANT PLASMA-CELL ,REPERTOIRE ,autologous stem cell transplantation ,ASO-RT-PCR ,BONE-MARROW TRANSPLANTATION ,myeloma clonally related cells - Abstract
A high incidence of oligoclonal serum M-components is observed in multiple myeloma (MM) patients treated with autologous Stem cell transplantation (ASCT). To determine whether these hi-components are produced by myeloma clonally related cells or caused by an aberrant B-cell regeneration we analysed by semi-nested ASO-RT-PCR and DNA sequencing the immunoglobulin (Ig) variable genes (VH) obtained from bone marrow samples obtained before and after transplantation and peripheral blood stern cell (PBSC) samples from seven patients. Myeloma clonally related cells are identifiable by the expression of variant Ig heavy chain isotypes and were detected in two patients at presentation. No myeloma clonally related cells were found in post-transplantation samples (n = 7) in spite of the appearance of new serum M-components, However, in two cases we amplified sequences from post-transplantation bone marrow cells that were able to bind to the B-cell clone-specific CDR3 oligonucleotides but showed no further similarity regarding the VDT rearrangement, These data indicate that serum oligoclonality posttransplantation is not caused by myeloma clonally related B cells but rather by the regenerating B-cell compartment.
- Published
- 1999
7. Structure and consequences of IGH switch breakpoints in Burkitt lymphoma.
- Author
-
Guikema JEJ, Schuuring E, and Kluin PM
- Published
- 2008
8. RAG1/2 induces double-stranded DNA breaks at non-Ig loci in the proximity of single sequence repeats in developing B cells.
- Author
-
Ochodnicka-Mackovicova K, Mokry M, Haagmans M, Bradley TE, van Noesel CJM, and Guikema JEJ
- Abstract
In developing B cells, V(D)J gene recombination is initiated by the RAG1/2 endonuclease complex, introducing double-stranded DNA breaks (DSBs) in V, D, and J genes and resulting in the formation of the hypervariable parts of immunoglobulins (Ig). Persistent or aberrant RAG1/2 targeting is a potential threat to genome integrity. While RAG1 and RAG2 have been shown to bind various regions genome-wide, the in vivo off-target DNA damage instigated by RAG1/2 endonuclease remains less well understood. In the current study, we identified regions containing RAG1/2-induced DNA breaks in mouse pre-B cells on a genome-wide scale using a global DNA DSB detection strategy. We detected 1489 putative RAG1/2-dependent DSBs, most of which were located outside the Ig loci. DNA sequence motif analysis showed a specific enrichment of RAG1/2-induced DNA DSBs at GA- and CA-repeats and GC-rich motifs. These findings provide further insights into RAG1/2 off-target activity. The ability of RAG1/2 to introduce DSBs on the non-Ig loci during the endogenous V(D)J recombination emphasizes its genotoxic potential in developing lymphocytes., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)
- Published
- 2024
- Full Text
- View/download PDF
9. Core regions in immunoglobulin heavy chain enhancers essential for survival of non-Hodgkin lymphoma cells are identified by a CRISPR interference screen.
- Author
-
Kasprzyk ME, Sura W, Podralska M, Kazimierska M, Seitz A, Łosiewski W, Woźniak T, Guikema JEJ, Diepstra A, Kluiver J, Van den Berg A, Rozwadowska N, and Dzikiewicz-Krawczyk A
- Abstract
Chromosomal translocations in non-Hodgkin lymphoma (NHL) result in activation of oncogenes by placing them under the regulation of immunoglobulin heavy chain (IGH) super-enhancers. Aberrant expression of translocated oncogenes induced by enhancer activity can contribute to lymphomagenesis. The role of the IGH enhancers in normal B-cell development is well established, but knowledge regarding the precise mechanisms of their involvement in control of the translocated oncogenes is limited. The goal of this project was to define the critical regions in the IGH regulatory elements and identify enhancer RNAs (eRNA). We designed a sgRNA library densely covering the IGH enhancers and performed tiling CRISPR interference screens in three NHL cell lines. This revealed three regions crucial for NHL cell growth. With chromatin-enriched RNA-Seq we showed transcription from the core enhancer regions and subsequently validated expression of the eRNAs in a panel of NHL cell lines and tissue samples. Inhibition of the essential IGH enhancer regions decreased expression of eRNAs and translocated oncogenes in several NHL cell lines. The observed expression and growth patterns were consistent with the breakpoints in the IGH locus. Moreover, targeting the Eμ enhancer resulted in loss of B-cell receptor expression. In a Burkitt lymphoma cell line, MYC overexpression partially rescued the phenotype induced by IGH enhancer inhibition. Our results indicated the most critical regions in the IGH enhancers and provided new insights into the current understanding of the role of IGH enhancers in B-cell NHL. As such, this study forms a basis for development of potential therapeutic approaches.
- Published
- 2024
- Full Text
- View/download PDF
10. Systematic evaluation of B-cell clonal family inference approaches.
- Author
-
Balashova D, van Schaik BDC, Stratigopoulou M, Guikema JEJ, Caniels TG, Claireaux M, van Gils MJ, Musters A, Anang DC, de Vries N, Greiff V, and van Kampen AHC
- Subjects
- Humans, Mutation, High-Throughput Nucleotide Sequencing, B-Lymphocytes, Receptors, Antigen, B-Cell genetics
- Abstract
The reconstruction of clonal families (CFs) in B-cell receptor (BCR) repertoire analysis is a crucial step to understand the adaptive immune system and how it responds to antigens. The BCR repertoire of an individual is formed throughout life and is diverse due to several factors such as gene recombination and somatic hypermutation. The use of Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using next generation sequencing enabled the generation of full BCR repertoires that also include rare CFs. The reconstruction of CFs from AIRR-seq data is challenging and several approaches have been developed to solve this problem. Currently, most methods use the heavy chain (HC) only, as it is more variable than the light chain (LC). CF reconstruction options include the definition of appropriate sequence similarity measures, the use of shared mutations among sequences, and the possibility of reconstruction without preliminary clustering based on V- and J-gene annotation. In this study, we aimed to systematically evaluate different approaches for CF reconstruction and to determine their impact on various outcome measures such as the number of CFs derived, the size of the CFs, and the accuracy of the reconstruction. The methods were compared to each other and to a method that groups sequences based on identical junction sequences and another method that only determines subclones. We found that after accounting for data set variability, in particular sequencing depth and mutation load, the reconstruction approach has an impact on part of the outcome measures, including the number of CFs. Simulations indicate that unique junctions and subclones should not be used as substitutes for CF and that more complex methods do not outperform simpler methods. Also, we conclude that different approaches differ in their ability to correctly reconstruct CFs when not considering the LC and to identify shared CFs. The results showed the effect of different approaches on the reconstruction of CFs and highlighted the importance of choosing an appropriate method., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
11. Levamisole suppresses activation and proliferation of human T cells by the induction of a p53-dependent DNA damage response.
- Author
-
Khan GH, Veltkamp F, Scheper M, Hoebe RA, Claessen N, Butter L, Bouts AHM, Florquin S, and Guikema JEJ
- Subjects
- Humans, Cell Division, Apoptosis, T-Lymphocytes, DNA Damage, Levamisole pharmacology, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Protein p53 metabolism
- Abstract
Levamisole (LMS) is a small molecule used in the treatment of idiopathic nephrotic syndrome (INS). The pathogenesis of INS remains unknown, but evidence points toward an immunological basis of the disease. Recently, LMS has been shown to increase the relapse-free survival in INS patients. While LMS has been hypothesized to exert an immunomodulatory effect, its mechanism of action remains unknown. Here, we show that LMS decreased activation and proliferation of human T cells. T-cell activation-associated cytokines such as IL-2, TNF-α, and IFN-γ were reduced upon LMS treatment, whereas IL-4 and IL-13 were increased. Gene expression profiling confirmed that the suppressive effects of LMS as genes involved in cell cycle progression were downregulated. Furthermore, genes associated with p53 activation were upregulated by LMS. In agreement, LMS treatment resulted in p53 phosphorylation and increased expression of the p53 target gene FAS. Accordingly, LMS sensitized activated T cells for Fas-mediated apoptosis. LMS treatment resulted in a mid-S phase cell cycle arrest accompanied by γH2AX-foci formation and phosphorylation of CHK1. Our findings indicate that LMS acts as an immunosuppressive drug that directly affects the activation and proliferation of human T cells by induction of DNA damage and the activation of a p53-dependent DNA damage response., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)
- Published
- 2023
- Full Text
- View/download PDF
12. AKT supports the metabolic fitness of multiple myeloma cells by restricting FOXO activity.
- Author
-
Bloedjes TA, de Wilde G, Khan GH, Ashby TC, Shaughnessy JD, Zhan F, Houtkooper RH, Bende RJ, van Noesel CJM, Spaargaren M, and Guikema JEJ
- Subjects
- Humans, Forkhead Transcription Factors genetics, Forkhead Transcription Factors metabolism, Oxidative Phosphorylation, Proto-Oncogene Proteins c-akt metabolism, Multiple Myeloma genetics
- Abstract
Metabolic alterations are important cancer-associated features that allow cancer cell transformation and survival under stress conditions. Multiple myeloma (MM) plasma cells show increased glycolysis and oxidative phosphorylation (OXPHOS), which are characteristics associated with recurrent genetic aberrations that drive the proliferation and survival of MM cells. The protein kinase B/AKT acts as a central node in cellular metabolism and is constitutively active in MM cells. Despite the known role of AKT in modulating cellular metabolism, little is known about the downstream factors of AKT that control the metabolic adaptability of MM cells. Here, we demonstrate that negative regulation of the forkhead box O (FOXO) transcription factors (TFs) by AKT is crucial to prevent the metabolic shutdown in MM cells, thus contributing to their metabolic adaptability. Our results demonstrate that the expression of several key metabolic genes involved in glycolysis, the tricarboxylic acid (TCA) cycle, and OXPHOS are repressed by FOXO TFs. Moreover, the FOXO-dependent repression of glycolysis- and TCA-associated genes correlates with a favorable prognosis in a large cohort of patients with MM. Our data suggest that repression of FOXO by AKT is essential to sustain glycolysis and the TCA cycle activity in MM cells and, as such, predicts patient survival., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
13. Exploring the impact of clonal definition on B-cell diversity: implications for the analysis of immune repertoires.
- Author
-
Pelissier A, Luo S, Stratigopoulou M, Guikema JEJ, and Rodríguez Martínez M
- Subjects
- Clone Cells, Immunoglobulins genetics, Gene Library, B-Lymphocytes, Receptors, Antigen, B-Cell
- Abstract
The adaptive immune system has the extraordinary ability to produce a broad range of immunoglobulins that can bind a wide variety of antigens. During adaptive immune responses, activated B cells duplicate and undergo somatic hypermutation in their B-cell receptor (BCR) genes, resulting in clonal families of diversified B cells that can be related back to a common ancestor. Advances in high-throughput sequencing technologies have enabled the high-throughput characterization of B-cell repertoires, however, the accurate identification of clonally related BCR sequences remains a major challenge. In this study, we compare three different clone identification methods on both simulated and experimental data, and investigate their impact on the characterization of B-cell diversity. We observe that different methods lead to different clonal definitions, which affects the quantification of clonal diversity in repertoire data. Our analyses show that direct comparisons between clonal clusterings and clonal diversity of different repertoires should be avoided if different clone identification methods were used to define the clones. Despite this variability, the diversity indices inferred from the repertoires' clonal characterization across samples show similar patterns of variation regardless of the clonal identification method used. We find the Shannon entropy to be the most robust in terms of the variability of diversity rank across samples. Our analysis also suggests that the traditional germline gene alignment-based method for clonal identification remains the most accurate when the complete information about the sequence is known, but that alignment-free methods may be preferred for shorter sequencing read lengths. We make our implementation freely available as a Python library cdiversity., Competing Interests: Authors AP, SL, and MR were employed by IBM Research Europe during this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pelissier, Luo, Stratigopoulou, Guikema and Rodríguez Martínez.)
- Published
- 2023
- Full Text
- View/download PDF
14. Understanding repertoire sequencing data through a multiscale computational model of the germinal center.
- Author
-
García-Valiente R, Merino Tejero E, Stratigopoulou M, Balashova D, Jongejan A, Lashgari D, Pélissier A, Caniels TG, Claireaux MAF, Musters A, van Gils MJ, Rodríguez Martínez M, de Vries N, Meyer-Hermann M, Guikema JEJ, Hoefsloot H, and van Kampen AHC
- Subjects
- Receptors, Antigen, B-Cell genetics, Germinal Center, B-Lymphocytes
- Abstract
Sequencing of B-cell and T-cell immune receptor repertoires helps us to understand the adaptive immune response, although it only provides information about the clonotypes (lineages) and their frequencies and not about, for example, their affinity or antigen (Ag) specificity. To further characterize the identified clones, usually with special attention to the particularly abundant ones (dominant), additional time-consuming or expensive experiments are generally required. Here, we present an extension of a multiscale model of the germinal center (GC) that we previously developed to gain more insight in B-cell repertoires. We compare the extent that these simulated repertoires deviate from experimental repertoires established from single GCs, blood, or tissue. Our simulations show that there is a limited correlation between clonal abundance and affinity and that there is large affinity variability among same-ancestor (same-clone) subclones. Our simulations suggest that low-abundance clones and subclones, might also be of interest since they may have high affinity for the Ag. We show that the fraction of plasma cells (PCs) with high B-cell receptor (BcR) mRNA content in the GC does not significantly affect the number of dominant clones derived from single GCs by sequencing BcR mRNAs. Results from these simulations guide data interpretation and the design of follow-up experiments., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
15. Inhibition of casein kinase 2 sensitizes mantle cell lymphoma to venetoclax through MCL-1 downregulation.
- Author
-
Thus YJ, De Rooij MFM, Swier N, Beijersbergen RL, Guikema JEJ, Kersten MJ, Eldering E, Pals ST, Kater AP, and Spaargaren M
- Subjects
- Humans, Adult, Myeloid Cell Leukemia Sequence 1 Protein metabolism, Casein Kinase II genetics, Casein Kinase II metabolism, Down-Regulation, Cell Line, Tumor, Proto-Oncogene Proteins c-bcl-2, Bridged Bicyclo Compounds, Heterocyclic pharmacology, Bridged Bicyclo Compounds, Heterocyclic therapeutic use, Lymphoma, Mantle-Cell drug therapy, Lymphoma, Mantle-Cell genetics, Lymphoma, Mantle-Cell metabolism, Antineoplastic Agents pharmacology, Antineoplastic Agents therapeutic use
- Abstract
BCL-2 family proteins are frequently aberrantly expressed in mantle cell lymphoma (MCL). Recently, the BCL-2-specific inhibitor venetoclax has been approved by the US Food and Drug Administration for chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). In MCL, venetoclax has shown promising efficacy in early clinical trials; however, a significant subset of patients is resistant. By conducting a kinome-centered CRISPR-Cas9 knockout sensitizer screen, we identified casein kinase 2 (CK2) as a major regulator of venetoclax resistance in MCL. Interestingly, CK2 is over-expressed in MCL and high CK2 expression is associated with poor patient survival. Targeting of CK2, either by inducible short hairpin RNA (shRNA)-mediated knockdown of CK2 or by the CK2-inhibitor silmitasertib, did not affect cell viability by itself, but strongly synergized with venetoclax in both MCL cell lines and primary samples, also if combined with ibrutinib. Furthermore, targeting of CK2 reduced MCL-1 levels, which involved impaired MCL-1 translation by inhibition of eIF4F complex assembly, without affecting BCL-2 and BCL-XL expression. Combined, this results in enhanced BCL-2 dependence and, consequently, venetoclax sensitization. In cocultures, targeting of CK2 overcame stroma-mediated venetoclax resistance of MCL cells. Taken together, our findings indicate that targeting of CK2 sensitizes MCL cells to venetoclax through downregulation of MCL-1. These novel insights provide a strong rationale for combining venetoclax with CK2 inhibition as therapeutic strategy for MCL patients.
- Published
- 2023
- Full Text
- View/download PDF
16. SMG1, a nonsense-mediated mRNA decay (NMD) regulator, as a candidate therapeutic target in multiple myeloma.
- Author
-
Leeksma AC, Derks IAM, Garrick B, Jongejan A, Colombo M, Bloedjes T, Trowe T, Leisten JC, Howarth M, Malek M, Mortensen DS, Blease K, Groza MC, Narla RK, Loos R, Kersten MJ, Moerland PD, Guikema JEJ, Kater AP, Eldering E, and Filvaroff EH
- Subjects
- Animals, Humans, Mice, Cell Line, DNA metabolism, Mammals genetics, Mammals metabolism, Protein Kinases metabolism, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Multiple Myeloma drug therapy, Multiple Myeloma genetics, Nonsense Mediated mRNA Decay genetics
- Abstract
Early data suggested that CC-115, a clinical molecule, already known to inhibit the mammalian target of rapamycin kinase (TORK) and DNA-dependent protein kinase (DNA-PK) may have additional targets beyond TORK and DNA-PK. Therefore, we aimed to identify such target(s) and investigate a potential therapeutic applicability. Functional profiling of 141 cancer cell lines revealed inhibition of kinase suppressor of morphogenesis in genitalia 1 (SMG1), a key regulator of the RNA degradation mechanism nonsense-mediated mRNA decay (NMD), as an additional target of CC-115. CC-115 treatment showed a dose-dependent increase of SMG1-mediated NMD transcripts. A subset of cell lines, including multiple myeloma (MM) cell lines sensitive to the endoplasmic reticulum stress-inducing compound thapsigargin, were highly susceptible to SMG1 inhibition. CC-115 caused the induction of UPR transcripts and cell death by mitochondrial apoptosis, requiring the presence of BAX/BAK and caspase activity. Superior antitumor activity of CC-115 over TORK inhibitors in primary human MM cells and three xenograft mouse models appeared to be via inhibition of SMG1. Our data support further development of SMG1 inhibitors as possible therapeutics in MM., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
- Published
- 2023
- Full Text
- View/download PDF
17. Huwe1 supports B-cell development, B-cell-dependent immunity, somatic hypermutation and class switch recombination by regulating proliferation.
- Author
-
Spanjaard A, Stratigopoulou M, de Groot D, Aslam M, van den Berk PCM, Stappenbelt C, Ayidah M, Catsman JJI, Pardieck IN, Kreft M, Arens R, Guikema JEJ, and Jacobs H
- Subjects
- B-Lymphocytes, DNA Repair, Cell Differentiation genetics, Somatic Hypermutation, Immunoglobulin, Immunoglobulin Class Switching genetics
- Abstract
The development and differentiation of B cells is intimately linked to cell proliferation and the generation of diverse immunoglobulin gene ( Ig ) repertoires. The ubiquitin E3 ligase HUWE1 controls proliferation, DNA damage responses, and DNA repair, including the base excision repair (BER) pathway. These processes are of crucial importance for B-cell development in the bone marrow, and the germinal center (GC) response, which results in the clonal expansion and differentiation of B cells expressing high affinity immunoglobulins. Here, we re-examined the role of HUWE1 in B-cell proliferation and Ig gene diversification, focusing on its involvement in somatic hypermutation (SHM) and class switch recombination (CSR). B-cell-specific deletion of Huwe1 resulted in impaired development, differentiation and maturation of B cells in the bone marrow and peripheral lymphoid organs. HUWE1 deficiency diminished SHM and CSR by impairing B-cell proliferation and AID expression upon activation in vitro and in vivo , and was unrelated to the HUWE1-dependent regulation of the BER pathway. Interestingly, we found that HUWE1-deficient B cells showed increased mRNA expression of Myc target genes upon in vitro activation despite diminished proliferation. Our results confirm that the E3 ligase HUWE1 is an important contributor in coordinating the rapid transition of antigen naïve, resting B cells into antigen-activated B cells and regulates mutagenic processes in B cells by controlling AID expression and the post-transcriptional output of Myc target genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Spanjaard, Stratigopoulou, de Groot, Aslam, van den Berk, Stappenbelt, Ayidah, Catsman, Pardieck, Kreft, Arens, Guikema and Jacobs.)
- Published
- 2023
- Full Text
- View/download PDF
18. Coupled Antigen and BLIMP1 Asymmetric Division With a Large Segregation Between Daughter Cells Recapitulates the Temporal Transition From Memory B Cells to Plasma Cells and a DZ-to-LZ Ratio in the Germinal Center.
- Author
-
Merino Tejero E, Lashgari D, García-Valiente R, He J, Robert PA, Meyer-Hermann M, Guikema JEJ, Hoefsloot H, and van Kampen AHC
- Subjects
- Biomarkers, Cell Differentiation, Gene Expression Regulation, Gene Regulatory Networks, Humans, Lymphocyte Activation, Memory B Cells metabolism, Models, Biological, Plasma Cells metabolism, Antigens immunology, Asymmetric Cell Division genetics, Germinal Center immunology, Germinal Center metabolism, Memory B Cells immunology, Plasma Cells immunology, Positive Regulatory Domain I-Binding Factor 1 genetics
- Abstract
Memory B cells and antibody-secreting plasma cells are generated within germinal centers during affinity maturation in which B-cell proliferation, selection, differentiation, and self-renewal play important roles. The mechanisms behind memory B cell and plasma cell differentiation in germinal centers are not well understood. However, it has been suggested that cell fate is (partially) determined by asymmetric cell division, which involves the unequal distribution of cellular components to both daughter cells. To investigate what level and/or probability of asymmetric segregation of several fate determinant molecules, such as the antigen and transcription factors (BCL6, IRF4, and BLIMP1) recapitulates the temporal switch and DZ-to-LZ ratio in the germinal center, we implemented a multiscale model that combines a core gene regulatory network for plasma cell differentiation with a model describing the cellular interactions and dynamics in the germinal center. Our simulations show that BLIMP1 driven plasma cell differentiation together with coupled asymmetric division of antigen and BLIMP1 with a large segregation between the daughter cells results in a germinal center DZ-to-LZ ratio and a temporal switch from memory B cells to plasma cells that have been observed in experiments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Merino Tejero, Lashgari, García-Valiente, He, Robert, Meyer-Hermann, Guikema, Hoefsloot and van Kampen.)
- Published
- 2021
- Full Text
- View/download PDF
19. A Major Subset of Mutated CLL Expresses Affinity-selected and Functionally Proficient Rheumatoid Factors.
- Author
-
Janssen J, Donner N, Li Z, Wormhoudt TAM, Wagner K, Guikema JEJ, van der Schoot CE, Kater AP, Feizi T, Bende RJ, and van Noesel CJM
- Published
- 2021
- Full Text
- View/download PDF
20. Multiscale Modeling of Germinal Center Recapitulates the Temporal Transition From Memory B Cells to Plasma Cells Differentiation as Regulated by Antigen Affinity-Based Tfh Cell Help.
- Author
-
Merino Tejero E, Lashgari D, García-Valiente R, Gao X, Crauste F, Robert PA, Meyer-Hermann M, Martínez MR, van Ham SM, Guikema JEJ, Hoefsloot H, and van Kampen AHC
- Subjects
- Asymmetric Cell Division, B-Lymphocytes cytology, Cell Lineage, Gene Regulatory Networks, Germinal Center cytology, Humans, Interferon Regulatory Factors genetics, Interferon Regulatory Factors physiology, Plasma Cells cytology, Positive Regulatory Domain I-Binding Factor 1 genetics, Positive Regulatory Domain I-Binding Factor 1 physiology, Proto-Oncogene Proteins c-bcl-6 genetics, Proto-Oncogene Proteins c-bcl-6 physiology, Signal Transduction, Time Factors, B-Lymphocytes immunology, CD40 Antigens immunology, Computer Simulation, Germinal Center immunology, Immunologic Memory immunology, Lymphopoiesis immunology, Models, Immunological, Plasma Cells immunology, T Follicular Helper Cells immunology
- Abstract
Germinal centers play a key role in the adaptive immune system since they are able to produce memory B cells and plasma cells that produce high affinity antibodies for an effective immune protection. The mechanisms underlying cell-fate decisions are not well understood but asymmetric division of antigen, B-cell receptor affinity, interactions between B-cells and T follicular helper cells (triggering CD40 signaling), and regulatory interactions of transcription factors have all been proposed to play a role. In addition, a temporal switch from memory B-cell to plasma cell differentiation during the germinal center reaction has been shown. To investigate if antigen affinity-based Tfh cell help recapitulates the temporal switch we implemented a multiscale model that integrates cellular interactions with a core gene regulatory network comprising BCL6, IRF4, and BLIMP1. Using this model we show that affinity-based CD40 signaling in combination with asymmetric division of B-cells result in switch from memory B-cell to plasma cell generation during the course of the germinal center reaction. We also show that cell fate division is unlikely to be (solely) based on asymmetric division of Ag but that BLIMP1 is a more important factor. Altogether, our model enables to test the influence of molecular modulations of the CD40 signaling pathway on the production of germinal center output cells., Competing Interests: MR was employed by the company IBM. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Merino Tejero, Lashgari, García-Valiente, Gao, Crauste, Robert, Meyer-Hermann, Martínez, van Ham, Guikema, Hoefsloot and van Kampen.)
- Published
- 2021
- Full Text
- View/download PDF
21. Metabolic Effects of Recurrent Genetic Aberrations in Multiple Myeloma.
- Author
-
Bloedjes TA, de Wilde G, and Guikema JEJ
- Abstract
Oncogene activation and malignant transformation exerts energetic, biosynthetic and redox demands on cancer cells due to increased proliferation, cell growth and tumor microenvironment adaptation. As such, altered metabolism is a hallmark of cancer, which is characterized by the reprogramming of multiple metabolic pathways. Multiple myeloma (MM) is a genetically heterogeneous disease that arises from terminally differentiated B cells. MM is characterized by reciprocal chromosomal translocations that often involve the immunoglobulin loci and a restricted set of partner loci, and complex chromosomal rearrangements that are associated with disease progression. Recurrent chromosomal aberrations in MM result in the aberrant expression of MYC, cyclin D1, FGFR3/MMSET and MAF/MAFB. In recent years, the intricate mechanisms that drive cancer cell metabolism and the many metabolic functions of the aforementioned MM-associated oncogenes have been investigated. Here, we discuss the metabolic consequences of recurrent chromosomal translocations in MM and provide a framework for the identification of metabolic changes that characterize MM cells.
- Published
- 2021
- Full Text
- View/download PDF
22. AKT signaling restrains tumor suppressive functions of FOXO transcription factors and GSK3 kinase in multiple myeloma.
- Author
-
Bloedjes TA, de Wilde G, Maas C, Eldering E, Bende RJ, van Noesel CJM, Pals ST, Spaargaren M, and Guikema JEJ
- Subjects
- Glycogen Synthase Kinase 3, Humans, Proto-Oncogene Proteins c-akt metabolism, Signal Transduction, Forkhead Transcription Factors metabolism, Multiple Myeloma drug therapy, Multiple Myeloma genetics
- Abstract
The phosphatidylinositide-3 kinases and the downstream mediator AKT drive survival and proliferation of multiple myeloma (MM) cells. AKT signaling is active in MM and has pleiotropic effects; however, the key molecular aspects of AKT dependency in MM are not fully clear. Among the various downstream AKT targets are the Forkhead box O (FOXO) transcription factors (TFs) and glycogen synthase kinase 3 (GSK3), which are negatively regulated by AKT signaling. Here we show that abrogation of AKT signaling in MM cells provokes cell death and cell cycle arrest, which crucially depends on both FOXO TFs and GSK3. Based on gene expression profiling, we defined a FOXO-repressed gene set that has prognostic significance in a large cohort of patients with MM, indicating that AKT-mediated gene activation is associated with inferior overall survival. We further show that AKT signaling stabilizes the antiapoptotic myeloid cell leukemia 1 (MCL1) protein by inhibiting FOXO- and GSK3-mediated MCL1 turnover. In concordance, abrogation of AKT signaling greatly sensitized MM cells for an MCL1-targeting BH3-mimetic, which is currently in clinical development. Taken together, our results indicate that AKT activity is required to restrain the tumor-suppressive functions of FOXO and GSK3, thereby stabilizing the antiapoptotic protein MCL1 in MM. These novel insights into the role of AKT in MM pathogenesis and MCL1 regulation provide opportunities to improve targeted therapy for patients with MM., (© 2020 by The American Society of Hematology.)
- Published
- 2020
- Full Text
- View/download PDF
23. Salivary Gland Mucosa-Associated Lymphoid Tissue-Type Lymphoma From Sjögren's Syndrome Patients in the Majority Express Rheumatoid Factors Affinity-Selected for IgG.
- Author
-
Bende RJ, Janssen J, Beentjes A, Wormhoudt TAM, Wagner K, Haacke EA, Kroese FGM, Guikema JEJ, and van Noesel CJM
- Subjects
- Humans, Lymphoma, B-Cell, Marginal Zone immunology, Receptors, Antigen, B-Cell genetics, Receptors, Antigen, B-Cell immunology, Salivary Glands immunology, Immunoglobulin G immunology, Lymphoma, B-Cell, Marginal Zone genetics, Mutation immunology, Rheumatoid Factor immunology, Sjogren's Syndrome genetics
- Abstract
Objective: Patients with Sjӧgren's syndrome (SS) have an increased risk of developing malignant B cell lymphomas, particularly mucosa-associated lymphoid tissue (MALT)-type lymphomas. We have previously shown that a predominant proportion of patients with SS-associated salivary gland MALT lymphoma express somatically hypermutated IgM with strong amino acid sequence homology with stereotypic rheumatoid factors (RFs). The present study was undertaken in a larger cohort of patients with SS-associated MALT lymphoma to more firmly assess the frequency of RF reactivity and the significance of somatic IGV-region mutations for RF reactivity., Methods: B cell antigen receptors (BCRs) of 16 patients with SS-associated salivary gland MALT lymphoma were analyzed. Soluble recombinant IgM was produced of 12 MALT lymphoma samples, including 1 MALT lymphoma sample that expressed an IgM antibody fitting in a novel IGHV3-30-encoded stereotypic IGHV subset. For 4 of the 12 IgM antibodies from MALT lymphoma samples, the somatically mutated IGHV and IGKV gene sequences were reverted to germline configurations. Their RF activity and binding affinity were determined by enzyme-linked immunosorbent assay and surface plasmon resonance, respectively., Results: Nine (75%) of the 12 IgM antibodies identified in patients with SS-associated salivary gland MALT lymphoma displayed strong monoreactive RF activity. Reversion of the IGHV and IGKV mutations to germline configuration resulted in RF affinities for IgG that were significantly lower for 3 of the 4 somatically mutated IgM antibodies. In stereotypic IGHV3-7/IGKV3-15-encoded RFs, a recurrent replacement mutation in the IGKV3-15-third complementarity-determining region was found to play a pivotal role in the affinity for IgG-Fc., Conclusion: A majority of patients with SS-associated salivary gland MALT lymphoma express somatically mutated BCRs that are selected for monoreactive, high-affinity binding of IgG-Fc. These data underscore the notion that soluble IgG, most likely in immune complexes in inflamed tissues, is the principal autoantigen in the pathogenesis of a variety of B cell lymphomas, particularly SS-associated MALT lymphomas., (© 2020 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)
- Published
- 2020
- Full Text
- View/download PDF
24. Base Excision Repair in the Immune System: Small DNA Lesions With Big Consequences.
- Author
-
Stratigopoulou M, van Dam TP, and Guikema JEJ
- Subjects
- Animals, DNA Breaks, Double-Stranded, DNA Damage, Humans, Immune System, Immunoglobulin Class Switching, Somatic Hypermutation, Immunoglobulin, B-Lymphocytes immunology, DNA genetics, DNA Repair immunology
- Abstract
The integrity of the genome is under constant threat of environmental and endogenous agents that cause DNA damage. Endogenous damage is particularly pervasive, occurring at an estimated rate of 10,000-30,000 per cell/per day, and mostly involves chemical DNA base lesions caused by oxidation, depurination, alkylation, and deamination. The base excision repair (BER) pathway is primary responsible for removing and repairing these small base lesions that would otherwise lead to mutations or DNA breaks during replication. Next to preventing DNA mutations and damage, the BER pathway is also involved in mutagenic processes in B cells during immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM), which are instigated by uracil (U) lesions derived from activation-induced cytidine deaminase (AID) activity. BER is required for the processing of AID-induced lesions into DNA double strand breaks (DSB) that are required for CSR, and is of pivotal importance for determining the mutagenic outcome of uracil lesions during SHM. Although uracils are generally efficiently repaired by error-free BER, this process is surprisingly error-prone at the Ig loci in proliferating B cells. Breakdown of this high-fidelity process outside of the Ig loci has been linked to mutations observed in B-cell tumors and DNA breaks and chromosomal translocations in activated B cells. Next to its role in preventing cancer, BER has also been implicated in immune tolerance. Several defects in BER components have been associated with autoimmune diseases, and animal models have shown that BER defects can cause autoimmunity in a B-cell intrinsic and extrinsic fashion. In this review we discuss the contribution of BER to genomic integrity in the context of immune receptor diversification, cancer and autoimmune diseases., (Copyright © 2020 Stratigopoulou, van Dam and Guikema.)
- Published
- 2020
- Full Text
- View/download PDF
25. The NEDD8-activating enzyme inhibitor MLN4924 induces DNA damage in Ph+ leukemia and sensitizes for ABL kinase inhibitors.
- Author
-
Bahjat M, de Wilde G, van Dam T, Maas C, Bloedjes T, Bende RJ, van Noesel CJM, Luijks DM, Eldering E, Kersten MJ, and Guikema JEJ
- Subjects
- Apoptosis drug effects, Cell Line, Tumor, Drug Synergism, Drug Therapy, Combination, Fusion Proteins, bcr-abl metabolism, Humans, Imatinib Mesylate pharmacology, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, Myeloid Cell Leukemia Sequence 1 Protein metabolism, Proto-Oncogene Proteins c-bcl-2 metabolism, S Phase Cell Cycle Checkpoints drug effects, Signal Transduction drug effects, Cyclopentanes pharmacology, DNA Damage drug effects, Drug Resistance, Neoplasm drug effects, Fusion Proteins, bcr-abl antagonists & inhibitors, Leukemia, Myelogenous, Chronic, BCR-ABL Positive metabolism, Protein Kinase Inhibitors pharmacology, Pyrimidines pharmacology, Ubiquitin-Activating Enzymes antagonists & inhibitors
- Abstract
The BCR-ABL1 fusion gene is the driver oncogene in chronic myeloid leukemia (CML) and Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). The introduction of tyrosine kinase inhibitors (TKIs) targeting the ABL kinase (such as imatinib) has dramatically improved survival of CML and Ph+ ALL patients. However, primary and acquired resistance to TKIs remains a clinical challenge. Ph+ leukemia patients who achieve a complete cytogenetic (CCR) or deep molecular response (MR) (≥4.5log reduction in BCR-ABL1 transcripts) represent long-term survivors. Thus, the fast and early eradication of leukemic cells predicts MR and is the prime clinical goal for these patients. We show here that the first-in-class inhibitor of the Nedd8-activating enzyme (NAE1) MLN4924 effectively induced caspase-dependent apoptosis in Ph+ leukemia cells, and sensitized leukemic cells for ABL tyrosine kinase inhibitors (TKI) and hydroxyurea (HU). We demonstrate that MLN4924 induced DNA damage in Ph+ leukemia cells by provoking the activation of an intra S-phase checkpoint, which was enhanced by imatinib co-treatment. The combination of MLN4924 and imatinib furthermore triggered a dramatic shift in the expression of MCL1 and NOXA. Our data offers a clear rationale to explore the clinical activity of MLN4924 (alone and in combination with ABL TKI) in Ph+ leukemia patients.
- Published
- 2019
- Full Text
- View/download PDF
26. De novo gene mutations in normal human memory B cells.
- Author
-
Slot LM, Wormhoudt TAM, Kwakkenbos MJ, Wagner K, Ballering A, Jongejan A, van Kampen ACM, Guikema JEJ, Bende RJ, and van Noesel CJM
- Subjects
- Clonal Selection, Antigen-Mediated, Humans, Immunoglobulin Heavy Chains genetics, Tetanus immunology, Exome Sequencing, B-Lymphocytes immunology, B-Lymphocytes metabolism, Immunologic Memory, Mutation
- Abstract
In the past years, the genomes of thousands of tumors have been elucidated. To date however, our knowledge on somatic gene alterations in normal cells is very limited. In this study, we demonstrate that tetanus-specific human memory B lymphocytes carry a substantial number of somatic mutations in the coding regions of the genome. Interestingly, we observed a statistically significant correlation between the number of exome mutations and those present in the immunoglobulin heavy variable regions. Our findings indicate that the majority of these genomic mutations arise in an antigen-dependent fashion, most likely during clonal expansion in germinal centers. The knowledge that normal B cells accumulate genomic alterations outside the immunoglobulin loci during development is relevant for our understanding of the process of lymphomagenesis.
- Published
- 2019
- Full Text
- View/download PDF
27. The Complex Interplay between DNA Injury and Repair in Enzymatically Induced Mutagenesis and DNA Damage in B Lymphocytes.
- Author
-
Bahjat M and Guikema JEJ
- Subjects
- B-Lymphocytes immunology, Cytidine Deaminase genetics, DNA Damage genetics, DNA Damage immunology, DNA Repair immunology, Gene Rearrangement genetics, Gene Rearrangement immunology, Humans, Mutagenesis genetics, Mutagenesis immunology, Somatic Hypermutation, Immunoglobulin immunology, T-Lymphocytes immunology, V(D)J Recombination immunology, DNA Repair genetics, Immunity, Humoral genetics, Somatic Hypermutation, Immunoglobulin genetics, V(D)J Recombination genetics
- Abstract
Lymphocytes are endowed with unique and specialized enzymatic mutagenic properties that allow them to diversify their antigen receptors, which are crucial sensors for pathogens and mediators of adaptive immunity. During lymphocyte development, the antigen receptors expressed by B and T lymphocytes are assembled in an antigen-independent fashion by ordered variable gene segment recombinations (V(D)J recombination), which is a highly ordered and regulated process that requires the recombination activating gene products 1 & 2 (RAG1, RAG2). Upon activation by antigen, B lymphocytes undergo additional diversifications of their immunoglobulin B-cell receptors. Enzymatically induced somatic hypermutation (SHM) and immunoglobulin class switch recombination (CSR) improves the affinity for antigen and shape the effector function of the humoral immune response, respectively. The activation-induced cytidine deaminase (AID) enzyme is crucial for both SHM and CSR. These processes have evolved to both utilize as well as evade different DNA repair and DNA damage response pathways. The delicate balance between enzymatic mutagenesis and DNA repair is crucial for effective immune responses and the maintenance of genomic integrity. Not surprisingly, disturbances in this balance are at the basis of lymphoid malignancies by provoking the formation of oncogenic mutations and chromosomal aberrations. In this review, we discuss recent mechanistic insight into the regulation of RAG1/2 and AID expression and activity in lymphocytes and the complex interplay between these mutagenic enzymes and DNA repair and DNA damage response pathways, focusing on the base excision repair and mismatch repair pathways. We discuss how disturbances of this interplay induce genomic instability and contribute to oncogenesis., Competing Interests: The authors declare that there is no conflict of interest.
- Published
- 2017
- Full Text
- View/download PDF
28. Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay.
- Author
-
Bahjat M, Bloedjes TA, van der Veen A, de Wilde G, Maas C, and Guikema JEJ
- Subjects
- DNA-Binding Proteins genetics, Humans, Precursor Cell Lymphoblastic Leukemia-Lymphoma metabolism, Cell Culture Techniques methods, DNA Damage, DNA-Activated Protein Kinase genetics, Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics
- Abstract
The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.
- Published
- 2017
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.