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Phosphoproteomic analysis of apoptotic hematopoietic stem cells from hemoglobin E/β-thalassemia.

Ponnikorn S, Panichakul T, Sresanga K, Wongborisuth C, Roytrakul S, Hongeng S, Tungpradabkul S - J Transl Med (2011)

Bottom Line: Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear.A significant change in abundance of 229 phosphoproteins was demonstrated.Importantly, the analysis of the candidate proteins revealed a high abundance of proteins that are commonly found in apoptotic cells including cytochrome C, caspase 6 and apoptosis inducing factors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.

ABSTRACT

Background: Hemoglobin E/β-thalassemia is particularly common in Southeast Asia and has variable symptoms ranging from mild to severe anemia. Previous investigations demonstrated the remarkable symptoms of β-thalassemia in terms of the acceleration of apoptotic cell death. Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear.

Methods: The phosphoproteome of bone marrow HSCs/CD34⁺ cells from HbE/β-thalassemic patients was analyzed using IMAC phosphoprotein isolation followed by LC-MS/MS detection. Decyder MS software was used to quantitate differentially expressed proteins in 3 patients and 2 normal donors. The differentially expressed proteins from HSCs/CD34⁺ cells were compared with HbE/β-thalassemia and normal HSCs.

Results: A significant change in abundance of 229 phosphoproteins was demonstrated. Importantly, the analysis of the candidate proteins revealed a high abundance of proteins that are commonly found in apoptotic cells including cytochrome C, caspase 6 and apoptosis inducing factors. Moreover, in the HSCs patients a significant increase was observed in a specific type of phosphoserine/threonine binding protein, which is known to act as an important signal mediator for the regulation of cell survival and apoptosis in HbE/β-thalassemia.

Conclusions: Our study used a novel method to investigate proteins that influence a particular pathway in a given disease or physiological condition. Ultimately, phosphoproteome profiling in HbE/β-thalassemic stem cells is an effective method to further investigate the cell death mechanism of ineffective erythropoiesis in β-thalassemia. Our report provides a comprehensive phosphoproteome, an important resource for the study of ineffective erythropoiesis and developing therapies for HbE/β-thalassemia.

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Cultures of HSC/CD34+ from HbE/β-thalassemic patients (A) and normal donors (B). After 7 days, the HbE/β thlassemic cell developed to erythroblasts and showed characteristic cell morphology of cells undergoing apoptosis including cell membrane blebbing and nuclear fragmentation.
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Figure 2: Cultures of HSC/CD34+ from HbE/β-thalassemic patients (A) and normal donors (B). After 7 days, the HbE/β thlassemic cell developed to erythroblasts and showed characteristic cell morphology of cells undergoing apoptosis including cell membrane blebbing and nuclear fragmentation.

Mentions: HSCs/CD34+ cells were isolated from 5 bone marrow samples belonging to 3 Hb E/β-thalassemic patients and 2 normal healthy donors. Flow cytometric analysis of confirmed that the isolated cells highly expressed the cell surface marker CD34 and had low levels of CD45 and were negative for glycophorin A. The purity of the isolated CD34+ cells was 92% (data not shown). CD34+ cells from patients and donors were cultured and cell viability was determined after 4 and 7 days of cultivation. The growth rate of thalassemic cells was reduced compared to normal cells (Figure 1). Giemsa staining revealed that CD34+ cells from patients and donors had similar morphological characteristics to blast cells with a large nucleus and 2-3 nucleoli. The characteristics of apoptotic cells, including membrane blebbling and nuclear fragmentation, were only found in thalassemic cells at days 4 and 7 (Figure 2).


Phosphoproteomic analysis of apoptotic hematopoietic stem cells from hemoglobin E/β-thalassemia.

Ponnikorn S, Panichakul T, Sresanga K, Wongborisuth C, Roytrakul S, Hongeng S, Tungpradabkul S - J Transl Med (2011)

Cultures of HSC/CD34+ from HbE/β-thalassemic patients (A) and normal donors (B). After 7 days, the HbE/β thlassemic cell developed to erythroblasts and showed characteristic cell morphology of cells undergoing apoptosis including cell membrane blebbing and nuclear fragmentation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3142509&req=5

Figure 2: Cultures of HSC/CD34+ from HbE/β-thalassemic patients (A) and normal donors (B). After 7 days, the HbE/β thlassemic cell developed to erythroblasts and showed characteristic cell morphology of cells undergoing apoptosis including cell membrane blebbing and nuclear fragmentation.
Mentions: HSCs/CD34+ cells were isolated from 5 bone marrow samples belonging to 3 Hb E/β-thalassemic patients and 2 normal healthy donors. Flow cytometric analysis of confirmed that the isolated cells highly expressed the cell surface marker CD34 and had low levels of CD45 and were negative for glycophorin A. The purity of the isolated CD34+ cells was 92% (data not shown). CD34+ cells from patients and donors were cultured and cell viability was determined after 4 and 7 days of cultivation. The growth rate of thalassemic cells was reduced compared to normal cells (Figure 1). Giemsa staining revealed that CD34+ cells from patients and donors had similar morphological characteristics to blast cells with a large nucleus and 2-3 nucleoli. The characteristics of apoptotic cells, including membrane blebbling and nuclear fragmentation, were only found in thalassemic cells at days 4 and 7 (Figure 2).

Bottom Line: Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear.A significant change in abundance of 229 phosphoproteins was demonstrated.Importantly, the analysis of the candidate proteins revealed a high abundance of proteins that are commonly found in apoptotic cells including cytochrome C, caspase 6 and apoptosis inducing factors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.

ABSTRACT

Background: Hemoglobin E/β-thalassemia is particularly common in Southeast Asia and has variable symptoms ranging from mild to severe anemia. Previous investigations demonstrated the remarkable symptoms of β-thalassemia in terms of the acceleration of apoptotic cell death. Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear.

Methods: The phosphoproteome of bone marrow HSCs/CD34⁺ cells from HbE/β-thalassemic patients was analyzed using IMAC phosphoprotein isolation followed by LC-MS/MS detection. Decyder MS software was used to quantitate differentially expressed proteins in 3 patients and 2 normal donors. The differentially expressed proteins from HSCs/CD34⁺ cells were compared with HbE/β-thalassemia and normal HSCs.

Results: A significant change in abundance of 229 phosphoproteins was demonstrated. Importantly, the analysis of the candidate proteins revealed a high abundance of proteins that are commonly found in apoptotic cells including cytochrome C, caspase 6 and apoptosis inducing factors. Moreover, in the HSCs patients a significant increase was observed in a specific type of phosphoserine/threonine binding protein, which is known to act as an important signal mediator for the regulation of cell survival and apoptosis in HbE/β-thalassemia.

Conclusions: Our study used a novel method to investigate proteins that influence a particular pathway in a given disease or physiological condition. Ultimately, phosphoproteome profiling in HbE/β-thalassemic stem cells is an effective method to further investigate the cell death mechanism of ineffective erythropoiesis in β-thalassemia. Our report provides a comprehensive phosphoproteome, an important resource for the study of ineffective erythropoiesis and developing therapies for HbE/β-thalassemia.

Show MeSH
Related in: MedlinePlus