p53 flow cytometry evaluation in leukemias: Correlation to factors affecting clinical outcome

Cavalcanti, Geraldo Barroso; Scheiner, Marcos Antônio Maurício; Magluta, Eliane Pereira Simões; Vasconcelos, Flavia da Cunha; Klumb, Claudete Esteves; Maia, Raquel Ciuvalschi

doi:10.1002/cyto.b.20514

Cited by 10 publications

(10 citation statements)

References 37 publications

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“…In mass cytometry, spillover is predictable and can thus be assessed and partly accounted for by designing appropriate panels and selecting appropriate antibody concentrations for downstream experiments [ 22 ]. Furthermore, the p53 (DO-7) antibody also detected p53 expression in the TP53 mutant cell lines NB4, OV90 and Raji, which is in concordance with previous reports [ 29 ].…”

Section: Resultssupporting

confidence: 92%

Single Cell Detection of the p53 Protein by Mass Cytometry

Fagerholt

Hellesøy

Gullaksen

et al. 2020

Cancers

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Purpose: The p53 protein and its post-translational modifications are distinctly expressed in various normal cell types and malignant cells and are usually detected by immunohistochemistry and flow cytometry in contemporary diagnostics. Here, we describe an approach for simultaneous multiparameter detection of p53, its post-translational modifications and p53 pathway-related signaling proteins in single cells using mass cytometry. Method: We conjugated p53-specific antibodies to metal tags for detection by mass cytometry, allowing the detection of proteins and their post-translational modifications in single cells. We provide an overview of the antibody validation process using relevant biological controls, including cell lines treated in vitro with a stimulus (irradiation) known to induce changes in the expression level of p53. Finally, we present the potential of the method through investigation of primary samples from leukemia patients with distinct TP53 mutational status. Results: The p53 protein can be detected in cell lines and in primary samples by mass cytometry. By combining antibodies for p53-related signaling proteins with a surface marker panel, we show that mass cytometry can be used to decipher the single cell p53 signaling pathway in heterogeneous patient samples. Conclusion: Single cell profiling by mass cytometry allows the investigation of the p53 functionality through examination of relevant downstream signaling proteins in normal and malignant cells. Our work illustrates a novel approach for single cell profiling of p53.

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Section: Resultssupporting

confidence: 92%

Single Cell Detection of the p53 Protein by Mass Cytometry

Fagerholt

Hellesøy

Gullaksen

et al. 2020

Cancers

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“…Therefore, our data demonstrated for the first time that the Jurkat/A4 cells with an acquired multidrug resistance phenotype are more sensitive to RPM microgravity, possibly due to the fact that Jurkat cells are a cancer p53-deficient cell line [46]. In our previous work, the results suggested that CD73 is one component of a complex nonspecific antiapoptotic program, which was, in our case, upregulated during selection for Fas resistance [27].…”

Section: Discussionsupporting

confidence: 55%

Changes in the Surface Expression of Intercellular Adhesion Molecule 3, the Induction of Apoptosis, and the Inhibition of Cell-Cycle Progression of Human Multidrug-Resistant Jurkat/A4 Cells Exposed to a Random Positioning Machine

Sokolovskaya

Korneeva

Zaichenko

et al. 2020

IJMS

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Experiments from flight- and ground-based model systems suggest that unexpected alterations of the human lymphoblastoid cell line Jurkat, as well as effects on cell growth, metabolism, and apoptosis, can occur in altered gravity conditions. Using a desktop random positioning machine (RPM), we investigated the effects of simulated microgravity on Jurkat cells and their multidrug-resistant subline, Jurkat/A4 cells. The viability of Jurkat/A4 cells decreased after simulated microgravity in contrast with the Jurkat cells. At the same time, the viability between the experimental Jurkat cells and control Jurkat cells was not significantly different. Of note, Jurkat cells appeared as less susceptible to apoptosis than their multidrug-resistant clone Jurkat/A4 cells, whereas cell-cycle analysis showed that the percentage of Jurkat/A4 cells in the S-phase was increased after 72 and 96 h of RPM-simulated microgravity relative to their static counterparts. The differences in Jurkat cells at all phases between static and simulated microgravity were not significant. The surface expression of the intercellular adhesion molecule 3 (ICAM-3)—also known as cluster of differentiation (CD)50—protein was changed for Jurkat/A4 cells following exposure to the RPM. Changes in cell morphology were observed in the Jurkat/A4 cells after 96 h of RPM-simulated microgravity. Thus, we concluded that Jurkat/A4 cells are more sensitive to RPM-simulated microgravity as compared with the parental Jurkat cell line. We also suggest that intercellular adhesion molecule 3 may be an important adhesion molecule involved in the induction of leukocyte apoptosis. The Jurkat/A4 cells with an acquired multidrug resistance phenotype could be a useful model for studying the effects of simulated microgravity and testing anticancer drugs.

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“…Our group detected p53 expression in samples from CML patients analysed by flow cytometry using anti-p53 monoclonal antibodies and an increased expression could be observed as the disease progressed to the blast phase ( Table 1 ). A positive relationship was found between p53 expression and high risk determined by Sokal score system, which is calculated using peripheral blood blast number, platelet count, spleen size and age in low-intermediate- or high-risk groups at the time of diagnosis [ 57 ].…”

Section: Introductionmentioning

confidence: 99%

Multidrug resistance in chronic myeloid leukaemia: how much can we learn from MDR–CML cell lines?

2013

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The hallmark of CML (chronic myeloid leukaemia) is the BCR (breakpoint cluster region)–ABL fusion gene. CML evolves through three phases, based on both clinical and pathological features: a chronic phase, an accelerated phase and blast crisis. TKI (tyrosine kinase inhibitors) are the treatment modality for patients with chronic phase CML. The therapeutic potential of the TKI imatinib is affected by BCR–ABL dependent an independent mechanisms. Development of MDR (multidrug resistance) contributes to the overall clinical resistance. MDR involves overexpression of ABC -transporters (ATP-binding-cassette transporter) among other features. MDR studies include the analysis of cancer cell lines selected for resistance. CML blast crisis is accompanied by increased resistance to apoptosis. This work reviews the role played by the influx transporter OCT1 (organic cation transporter 1), by efflux ABC transporters, molecules involved in the modulation of apoptosis (p53, Bcl-2 family, CD95, IAPs (inhibitors of apoptosis protein)], Hh and Wnt/β-catenin pathways, cytoskeleton abnormalities and other features described in leukaemic cells of clinical samples and CML cell lines. An MDR cell line, Lucena-1, generated from K562 by stepwise exposure to vincristine, was used as our model and some potential anticancer drugs effective against the MDR cell line and patients’ samples are presented.

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p53 flow cytometry evaluation in leukemias: Correlation to factors affecting clinical outcome

Cited by 10 publications

References 37 publications

Single Cell Detection of the p53 Protein by Mass Cytometry

Single Cell Detection of the p53 Protein by Mass Cytometry

Changes in the Surface Expression of Intercellular Adhesion Molecule 3, the Induction of Apoptosis, and the Inhibition of Cell-Cycle Progression of Human Multidrug-Resistant Jurkat/A4 Cells Exposed to a Random Positioning Machine

Multidrug resistance in chronic myeloid leukaemia: how much can we learn from MDR–CML cell lines?

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