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Peripheral blood cells from children with RASopathies show enhanced spontaneous colonies growth in vitro and hyperactive RAS signaling.

Gaipa G, Bugarin C, Cianci P, Sarno J, Bonaccorso P, Biondi A, Selicorni A - Blood Cancer J (2015)

Bottom Line: Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects.We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies.Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients.

View Article: PubMed Central - PubMed

Affiliation: M. Tettamanti Research Centre, Pediatric Clinic, University of Milano-Bicocca, Monza, Italy.

ABSTRACT
Germline mutations in genes coding for molecules involved in the RAS/RAF/MEK/ERK pathway are the hallmarks of a newly classified family of autosomal dominant syndromes termed RASopathies. Myeloproliferative disorders (MPDs), in particular, juvenile myelomonocytic leukemia, can lead to potentially severe complications in children with Noonan syndrome (NS). We studied 27 children with NS or other RASopathies and 35 age-matched children as control subjects. Peripheral blood (PB) cells from these patients were studied for in vitro colony-forming units (CFUs) activity, as well as for intracellular phosphosignaling. Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects. We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies. Our findings demonstrate for the first time that PB cells isolated from children suffering from NS or other RASopathies without MPD display enhanced BFU-E and CFU-GM colony formation in vitro. The biological significance of these findings clearly awaits further studies. Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients.

No MeSH data available.


Related in: MedlinePlus

Mononuclear cells were identified on the basis of physical parameters (a) and then selected according to their CD45 reactivity (b). Within the CD45+ cells, monocytes (in green), non-myeloid and myeloid cells could be distinguished by means of their CD33 and CD14 reactivity (c). Phosphoprotein expression was then measured on monocytic gated cells (d–f).
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fig1: Mononuclear cells were identified on the basis of physical parameters (a) and then selected according to their CD45 reactivity (b). Within the CD45+ cells, monocytes (in green), non-myeloid and myeloid cells could be distinguished by means of their CD33 and CD14 reactivity (c). Phosphoprotein expression was then measured on monocytic gated cells (d–f).

Mentions: Samples were stained with antibodies such as CD33 PE (clone P67.6), CD14 APC-H7 (clone MϕP9), CD45 PerCP (clone 2D1), p-STAT5 Alexa 647 (pY694), p-STAT3 Alexa 488 (pY705), p-ERK1/2 Alexa 488 (T202/Y204) or isotypes IgG1k Alexa 488 and Alexa 647 (clone MOPC-21) (BD Bioscience, San José, CA, USA), and data were acquired on a FACSaria flow cytometer (BD Bioscience). At least 100 000 events were collected and analyzed using DIVA software (San José, CA, USA). Basal levels of each phosphoprotein were calculated as percentage of phospho-positive cells in unstimulated conditions. Response to stimulation upon cytokine treatment (for example, recombinant human GM-CSF or IL-6) was calculated by subtracting the percentage of unstimulated phospho-positive cells under basal conditions. Each phosphoprotein reactivity was evaluated in CD14+/CD33+/CD45+ cells as assessed by the flow cytometric gating strategy described in Figure 1. GM-CSF-induced p-STAT5 signaling in monocytic cells contained was considered as an internal positive control of functional signaling.


Peripheral blood cells from children with RASopathies show enhanced spontaneous colonies growth in vitro and hyperactive RAS signaling.

Gaipa G, Bugarin C, Cianci P, Sarno J, Bonaccorso P, Biondi A, Selicorni A - Blood Cancer J (2015)

Mononuclear cells were identified on the basis of physical parameters (a) and then selected according to their CD45 reactivity (b). Within the CD45+ cells, monocytes (in green), non-myeloid and myeloid cells could be distinguished by means of their CD33 and CD14 reactivity (c). Phosphoprotein expression was then measured on monocytic gated cells (d–f).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526778&req=5

fig1: Mononuclear cells were identified on the basis of physical parameters (a) and then selected according to their CD45 reactivity (b). Within the CD45+ cells, monocytes (in green), non-myeloid and myeloid cells could be distinguished by means of their CD33 and CD14 reactivity (c). Phosphoprotein expression was then measured on monocytic gated cells (d–f).
Mentions: Samples were stained with antibodies such as CD33 PE (clone P67.6), CD14 APC-H7 (clone MϕP9), CD45 PerCP (clone 2D1), p-STAT5 Alexa 647 (pY694), p-STAT3 Alexa 488 (pY705), p-ERK1/2 Alexa 488 (T202/Y204) or isotypes IgG1k Alexa 488 and Alexa 647 (clone MOPC-21) (BD Bioscience, San José, CA, USA), and data were acquired on a FACSaria flow cytometer (BD Bioscience). At least 100 000 events were collected and analyzed using DIVA software (San José, CA, USA). Basal levels of each phosphoprotein were calculated as percentage of phospho-positive cells in unstimulated conditions. Response to stimulation upon cytokine treatment (for example, recombinant human GM-CSF or IL-6) was calculated by subtracting the percentage of unstimulated phospho-positive cells under basal conditions. Each phosphoprotein reactivity was evaluated in CD14+/CD33+/CD45+ cells as assessed by the flow cytometric gating strategy described in Figure 1. GM-CSF-induced p-STAT5 signaling in monocytic cells contained was considered as an internal positive control of functional signaling.

Bottom Line: Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects.We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies.Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients.

View Article: PubMed Central - PubMed

Affiliation: M. Tettamanti Research Centre, Pediatric Clinic, University of Milano-Bicocca, Monza, Italy.

ABSTRACT
Germline mutations in genes coding for molecules involved in the RAS/RAF/MEK/ERK pathway are the hallmarks of a newly classified family of autosomal dominant syndromes termed RASopathies. Myeloproliferative disorders (MPDs), in particular, juvenile myelomonocytic leukemia, can lead to potentially severe complications in children with Noonan syndrome (NS). We studied 27 children with NS or other RASopathies and 35 age-matched children as control subjects. Peripheral blood (PB) cells from these patients were studied for in vitro colony-forming units (CFUs) activity, as well as for intracellular phosphosignaling. Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects. We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies. Our findings demonstrate for the first time that PB cells isolated from children suffering from NS or other RASopathies without MPD display enhanced BFU-E and CFU-GM colony formation in vitro. The biological significance of these findings clearly awaits further studies. Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients.

No MeSH data available.


Related in: MedlinePlus