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1.
NPJ Precis Oncol ; 8(1): 107, 2024 May 20.
Article in English | MEDLINE | ID: mdl-38769096

ABSTRACT

The microenvironment of hematologic cancers contributes to tumor cell survival and proliferation, as well as treatment resistance. Understanding tumor- and drug-induced changes to the immune cell composition and functionality is therefore critical for implementing optimal treatment strategies and for the development of novel cancer therapies. The liquid nature of peripheral blood makes this organ uniquely suited for single-cell studies by flow cytometry. (Phospho)protein profiles detected by flow cytometry analyses have been shown to correlate with ex vivo drug sensitivity and to predict treatment outcomes in hematologic cancers, demonstrating that this method is suitable for pre-clinical studies. Here, we present a flow cytometry protocol that combines multi-parameter immunophenotyping with single-cell (phospho)protein profiling. The protocol makes use of fluorescent cell barcoding, which means that multiple cell samples, either collected from different donors or exposed to different treatment conditions, can be combined and analyzed as one experiment. This reduces variability between samples, increases the throughput of the experiment, and lowers experimental costs. This protocol may serve as a guide for the use and further development of assays to study immunophenotype and cell signaling at single-cell resolution in normal and malignant cells. The read-outs may provide biological insight into cancer pathogenesis, identify novel drug targets, and ultimately serve as a biomarker to guide clinical decision-making.

2.
Sci Rep ; 11(1): 6317, 2021 03 18.
Article in English | MEDLINE | ID: mdl-33737576

ABSTRACT

Chemo-immunotherapy has improved survival in B-cell lymphoma patients, but refractory/relapsed diseases still represent a major challenge, urging for development of new therapeutics. Karonudib (TH1579) was developed to inhibit MTH1, an enzyme preventing oxidized dNTP-incorporation in DNA. MTH1 is highly upregulated in tumor biopsies from patients with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma, hence confirming a rationale for targeting MTH1. Here, we tested the efficacy of karonudib in vitro and in preclinical B-cell lymphoma models. Using a range of B-cell lymphoma cell lines, karonudib strongly reduced viability at concentrations well tolerated by activated normal B cells. In B-cell lymphoma cells, karonudib increased incorporation of 8-oxo-dGTP into DNA, and prominently induced prometaphase arrest and apoptosis due to failure in spindle assembly. MTH1 knockout cell lines were less sensitive to karonudib-induced apoptosis, but were displaying cell cycle arrest phenotype similar to the wild type cells, indicating a dual inhibitory role of the drug. Karonudib was highly potent as single agent in two different lymphoma xenograft models, including an ABC DLBCL patient derived xenograft, leading to prolonged survival and fully controlled tumor growth. Together, our preclinical findings provide a rationale for further clinical testing of karonudib in B-cell lymphoma.


Subject(s)
Burkitt Lymphoma/drug therapy , DNA Repair Enzymes/genetics , Lymphoma, B-Cell/drug therapy , Phosphoric Monoester Hydrolases/genetics , Pyrimidines/pharmacology , Animals , Apoptosis/drug effects , Burkitt Lymphoma/genetics , Burkitt Lymphoma/pathology , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DNA/biosynthesis , DNA Repair Enzymes/antagonists & inhibitors , Deoxyguanine Nucleotides/antagonists & inhibitors , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/pathology , Mice , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Xenograft Model Antitumor Assays
3.
PLoS One ; 6(2): e17175, 2011 Feb 28.
Article in English | MEDLINE | ID: mdl-21386895

ABSTRACT

The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1) and G(2) phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase. We have devised a flow cytometric method exploiting the fact that cells in G(1) phase contain two nuclei, whereas cells in G(2) are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G(1) and in G(2) phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.


Subject(s)
Cell Cycle/physiology , Flow Cytometry , Schizosaccharomyces/cytology , Cell Cycle/genetics , Cell Cycle Proteins/genetics , Cell Proliferation , Flow Cytometry/methods , G1 Phase/genetics , Light , Mitosis/genetics , Mitosis/physiology , Models, Biological , Organisms, Genetically Modified , Scattering, Radiation , Schizosaccharomyces/growth & development , Schizosaccharomyces/physiology , Schizosaccharomyces pombe Proteins/genetics , Transcription Factors/genetics , ras-GRF1/genetics
4.
Cell Cycle ; 10(5): 819-29, 2011 Mar 01.
Article in English | MEDLINE | ID: mdl-21325885

ABSTRACT

Cell cycle checkpoints ensure that eukaryotic cells do not enter mitosis after ionizing irradiation (IR). The G(2)-arrest after IR is the result of activation of multiple signalling pathways, the contributions of which vary with time after irradiation. We have studied the time evolution of the IR-induced G(2)-arrest in human B-lymphocyte cancer cell lines, as well as the molecular mechanisms responsible for the arrest. Cells that were in G(2) phase at the time of irradiation experienced a transient arrest that blocked entry into mitosis at 0-2 hours after IR (0.5 or 4 Gy). Activation of ATM and CHEK2 occurred at the same time as this early arrest and was, like the arrest, abrogated by the ATM-inhibitor KU-55933. A late, permanent and ATM-independent arrest (≥6 hours after IR) of cells that were in G(2)/S/G(1) at the time of irradiation (4 Gy) was inactivated by caffeine. This late G(2)-arrest could not be explained by down-regulation of genes with functions in G(2)/mitosis (e.g. PLK1, CCNB1/2), since the down-regulation was transient and not accompanied by reduced protein levels. However, the persistent phosphorylation of CHEK1 after 4 Gy suggested a role for CHEK1 in the late arrest, consistent with the abrogation of the arrest in CHEK1-depleted cells. TP53 was not necessary for the late G(2)-arrest, but mediated an intermediate arrest (2-10 hours after IR) independently of ATM and CHEK1. In conclusion, the IR-induced arrest in G(2) is mediated by ATM immediately after irradiation, with TP53 for independent and transient back-up, while CHEK1 is necessary for the late arrest.


Subject(s)
G2 Phase/radiation effects , Radiation, Ionizing , Ataxia Telangiectasia Mutated Proteins , Caffeine/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Checkpoint Kinase 1 , Checkpoint Kinase 2 , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Humans , Mitosis , Morpholines/pharmacology , Phosphorylation , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Pyrones/pharmacology , RNA Interference , RNA, Small Interfering/metabolism , Signal Transduction , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/metabolism
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