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Updated: Jul 27, 2025
Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
Published on: April 24, 2011
Nucleolar phosphoprotein modifications as a marker of apoptosis induced by RITA treatment
Kateřina Wolfová1, Petra Otevřelová1, Aleš Holoubek1
1Department of Proteomics, Institute of Hematology and Blood Transfusion, U Nemocnice 1, 12820 Prague 2, Czech Republic.
Abstract:
Reactivating p53 and Inducing Tumor Apoptosis (RITA) has been reported to increase the p53 activity and to trigger p53-dependent apoptosis in cancer cells with wild-type p53. Tumor suppressor p53 interacts with nucleolar phosphoproteins nucleophosmin (NPM) and nucleolin (NCL), which have crucial role in many cellular processes. Specific NPM mutations associated with acute myeloid leukemia (AML) cause aberrant localization of NPM and p53 in the cytoplasm with possible impact on the p53 function. We tested an effect of RITA on primary cells, and we found significant RITA-induced changes in NPM and NCL phosphorylation associated with apoptosis in cells of AML patients, but not that of healthy donors. Subsequent screening of several AML cell lines revealed heterogeneous response to RITA, and confirmed an association of the specific phosphorylation with apoptosis. While decreased NCL phosphorylation at Threonines T76 and T84 could be attributed to RITA-induced cell cycle arrest, enhanced NPM phosphorylation at Threonine T199 was not accompanied by the cell cycle changes and it correlated with sensitivity to RITA. Simultaneously, inverse changes occurred at Serine S4 of the NPM. These new findings of RITA mechanism of action could establish the NPM pT199/pS4 ratio as a marker for suitability of RITA treatment of AML cells.
Insights
Reactivating p53 and Inducing Tumor Apoptosis (RITA) triggers apoptosis in acute myeloid leukemia (AML) cells by altering nucleophosmin (NPM) and nucleolin (NCL) phosphorylation. The NPM pT199/pS4 ratio may predict RITA treatment suitability.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- The tumor suppressor p53 plays a critical role in cancer, interacting with nucleolar phosphoproteins nucleophosmin (NPM) and nucleolin (NCL).
- Mutations in NPM in acute myeloid leukemia (AML) can lead to aberrant cytoplasmic localization of NPM and p53, potentially affecting p53 function.
- Reactivating p53 and Inducing Tumor Apoptosis (RITA) is a compound that enhances p53 activity and induces apoptosis in cancer cells with wild-type p53.
Purpose of the Study:
- To investigate the effect of RITA on NPM and NCL phosphorylation in primary cells from AML patients and healthy donors.
- To screen AML cell lines for heterogeneous responses to RITA and identify specific phosphorylation patterns associated with apoptosis.
- To explore the potential of NPM phosphorylation patterns as predictive markers for RITA treatment efficacy in AML.
Main Methods:
- Treatment of primary cells from AML patients and healthy donors with RITA.
- Screening of various AML cell lines for their response to RITA.
- Analysis of NPM and NCL phosphorylation status, specifically at Threonines T76 and T84 for NCL, and Threonine T199 and Serine S4 for NPM.
- Assessment of RITA-induced apoptosis and cell cycle changes.
Main Results:
- RITA induced significant changes in NPM and NCL phosphorylation, correlating with apoptosis in AML cells but not in healthy donor cells.
- A heterogeneous response to RITA was observed across different AML cell lines.
- Decreased NCL phosphorylation at T76 and T84 was linked to RITA-induced cell cycle arrest.
- Enhanced NPM phosphorylation at T199, inversely correlated with NPM phosphorylation at S4, was associated with RITA sensitivity and not cell cycle arrest.
Conclusions:
- RITA treatment significantly impacts NPM and NCL phosphorylation in AML cells, leading to apoptosis.
- The NPM phosphorylation ratio of pT199/pS4 emerges as a potential predictive biomarker for RITA treatment response in AML.
- These findings elucidate a novel mechanism of RITA action and offer a potential strategy for personalized AML therapy.
