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Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera.

Gallardo M, Barrio S, Fernandez M, Paradela A, Arenas A, Toldos O, Ayala R, Albizua E, Jimenez A, Redondo S, Garcia-Martin RM, Gilsanz F, Albar JP, Martinez-Lopez J - Mol. Cancer (2013)

Bottom Line: Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression.In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70.These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hematology Service, Hospital Universitario 12 de Octubre, Avenida, Córdoba, s/n, 28041, Madrid, Spain. miguelgallardodelgado@gmail.com.

ABSTRACT
JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

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Two-dimensional difference gel electrophoresis (2D-DIGE) analysis and IHC validation. (A) Representative spots from three proteins from 2D-DIGE gels and intensity quantification with DeCyder software v7.0. three spots were identified as LTA4H, SERPINB1 and HSP70 (HSPA1A). These spots were over-expressed in polycythemia vera (PV) vs. essential thrombocythemia (ET) samples. (B) Representative images according the median of HSP70 staining of granulocytes from bone marrow of PV, and ET. (C) Box-plot of percentage of HSP70 positive granulocytes quantified from IHC.
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Figure 2: Two-dimensional difference gel electrophoresis (2D-DIGE) analysis and IHC validation. (A) Representative spots from three proteins from 2D-DIGE gels and intensity quantification with DeCyder software v7.0. three spots were identified as LTA4H, SERPINB1 and HSP70 (HSPA1A). These spots were over-expressed in polycythemia vera (PV) vs. essential thrombocythemia (ET) samples. (B) Representative images according the median of HSP70 staining of granulocytes from bone marrow of PV, and ET. (C) Box-plot of percentage of HSP70 positive granulocytes quantified from IHC.

Mentions: DIGE and MS were used to identify differences in the whole cytosolic proteome between PV and ET groups. Figure 2A, show three representative spots from the proteomic analyses of samples from ET and PV patients. We found 112 spots representing proteins with differential expression between both diseases. Identification of the spots yielded 65 proteins. Three proteins were especially interesting in the context of our model and selected for further studies by doing a literature search on their biological function. These three differentially expressed proteins includes LTA4H, SERPINB1 and HSP70 (Figure 2A). Of note, HSP70 is a chaperone related to GATA-1 and erythroid differentiation. Most of the other spots corresponded to a large group of proteins implicated in metabolic and biochemical processes, for example, glycogen phosphorylase, pyruvate kinase, and lactotransferrin. Healthy donors also showed differences when compared with PV samples. There were 174 spots and 19 proteins identified (HSP70 included). Samples from controls and ET showed differences in 97 spots, and six proteins were identified. Most of the proteins identified were implicated in metabolic and biochemical pathways, similarly to those observed when ET and PV were compared. A full list of the differentially expressed proteins is summarized in Additional file 1: Table S1, Additional file 2: Table S2 and Additional file 3: Table S3.


Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera.

Gallardo M, Barrio S, Fernandez M, Paradela A, Arenas A, Toldos O, Ayala R, Albizua E, Jimenez A, Redondo S, Garcia-Martin RM, Gilsanz F, Albar JP, Martinez-Lopez J - Mol. Cancer (2013)

Two-dimensional difference gel electrophoresis (2D-DIGE) analysis and IHC validation. (A) Representative spots from three proteins from 2D-DIGE gels and intensity quantification with DeCyder software v7.0. three spots were identified as LTA4H, SERPINB1 and HSP70 (HSPA1A). These spots were over-expressed in polycythemia vera (PV) vs. essential thrombocythemia (ET) samples. (B) Representative images according the median of HSP70 staining of granulocytes from bone marrow of PV, and ET. (C) Box-plot of percentage of HSP70 positive granulocytes quantified from IHC.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225507&req=5

Figure 2: Two-dimensional difference gel electrophoresis (2D-DIGE) analysis and IHC validation. (A) Representative spots from three proteins from 2D-DIGE gels and intensity quantification with DeCyder software v7.0. three spots were identified as LTA4H, SERPINB1 and HSP70 (HSPA1A). These spots were over-expressed in polycythemia vera (PV) vs. essential thrombocythemia (ET) samples. (B) Representative images according the median of HSP70 staining of granulocytes from bone marrow of PV, and ET. (C) Box-plot of percentage of HSP70 positive granulocytes quantified from IHC.
Mentions: DIGE and MS were used to identify differences in the whole cytosolic proteome between PV and ET groups. Figure 2A, show three representative spots from the proteomic analyses of samples from ET and PV patients. We found 112 spots representing proteins with differential expression between both diseases. Identification of the spots yielded 65 proteins. Three proteins were especially interesting in the context of our model and selected for further studies by doing a literature search on their biological function. These three differentially expressed proteins includes LTA4H, SERPINB1 and HSP70 (Figure 2A). Of note, HSP70 is a chaperone related to GATA-1 and erythroid differentiation. Most of the other spots corresponded to a large group of proteins implicated in metabolic and biochemical processes, for example, glycogen phosphorylase, pyruvate kinase, and lactotransferrin. Healthy donors also showed differences when compared with PV samples. There were 174 spots and 19 proteins identified (HSP70 included). Samples from controls and ET showed differences in 97 spots, and six proteins were identified. Most of the proteins identified were implicated in metabolic and biochemical pathways, similarly to those observed when ET and PV were compared. A full list of the differentially expressed proteins is summarized in Additional file 1: Table S1, Additional file 2: Table S2 and Additional file 3: Table S3.

Bottom Line: Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression.In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70.These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hematology Service, Hospital Universitario 12 de Octubre, Avenida, Córdoba, s/n, 28041, Madrid, Spain. miguelgallardodelgado@gmail.com.

ABSTRACT
JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

Show MeSH
Related in: MedlinePlus