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Characterization of ectonucleotidases in human medulloblastoma cell lines: ecto-5'NT/CD73 in metastasis as potential prognostic factor.

Cappellari AR, Rockenbach L, Dietrich F, Clarimundo V, Glaser T, Braganhol E, Abujamra AL, Roesler R, Ulrich H, Battastini AM, Battastini AM - PLoS ONE (2012)

Bottom Line: On the other hand, D283 cell line did not hydrolyze AMP.It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity.In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas e da Saúde, UFRGS, Porto Alegre, Rio Grande do Sol, Brasil.

ABSTRACT
Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5'NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5'NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5'NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

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Cytosolic and nuclear P-β-catenin expression in MB cell lines.Phospho-β-catenin expression was determined by (A) Immunofluorescence and (B) Western blotting assays as described in Materials and Methods. For immunofluorescence, images were captured at objective lens of 20×magnification and the cytosolic and nuclear Phospho-β-catenin immunoreactivity were observed at merge column. For Western blotting, relative Phospho-β-catenin levels of the cytosolic and nuclear fractions obtained by densimetric analysis of protein bands detected in Western blots were compared to GAPDH and HnRNPK expression levels, respectively. Bars represent mean ± SD of 3 independent experiments performed in triplicate. Data were compared by One-Way ANOVA test following by the Tukey post hoc test. (* p<0,01) compared to control data.
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pone-0047468-g009: Cytosolic and nuclear P-β-catenin expression in MB cell lines.Phospho-β-catenin expression was determined by (A) Immunofluorescence and (B) Western blotting assays as described in Materials and Methods. For immunofluorescence, images were captured at objective lens of 20×magnification and the cytosolic and nuclear Phospho-β-catenin immunoreactivity were observed at merge column. For Western blotting, relative Phospho-β-catenin levels of the cytosolic and nuclear fractions obtained by densimetric analysis of protein bands detected in Western blots were compared to GAPDH and HnRNPK expression levels, respectively. Bars represent mean ± SD of 3 independent experiments performed in triplicate. Data were compared by One-Way ANOVA test following by the Tukey post hoc test. (* p<0,01) compared to control data.

Mentions: β-catenin is a downstream effector of the Wnt canonical pathway and, as described before, it is important for MB tumor prognosis and staging [3], [5]. We used immunofluorescence and Western blotting assays for detecting phospho-β-catenin expression in the MB cell lines used here. Firstly, we observed that Daoy and ONS76 showed mainly nuclear immunoreactivity to phospho-β-catenin, while the D283 MB cell line revealed immunoreactivity for this protein in the cytosol (Figure 9A). These qualitative data were confirmed by Western blotting of cytosolic and nuclear fractions compared to their respective controls, GAPDH and HnRNPK (Figure 9B).


Characterization of ectonucleotidases in human medulloblastoma cell lines: ecto-5'NT/CD73 in metastasis as potential prognostic factor.

Cappellari AR, Rockenbach L, Dietrich F, Clarimundo V, Glaser T, Braganhol E, Abujamra AL, Roesler R, Ulrich H, Battastini AM, Battastini AM - PLoS ONE (2012)

Cytosolic and nuclear P-β-catenin expression in MB cell lines.Phospho-β-catenin expression was determined by (A) Immunofluorescence and (B) Western blotting assays as described in Materials and Methods. For immunofluorescence, images were captured at objective lens of 20×magnification and the cytosolic and nuclear Phospho-β-catenin immunoreactivity were observed at merge column. For Western blotting, relative Phospho-β-catenin levels of the cytosolic and nuclear fractions obtained by densimetric analysis of protein bands detected in Western blots were compared to GAPDH and HnRNPK expression levels, respectively. Bars represent mean ± SD of 3 independent experiments performed in triplicate. Data were compared by One-Way ANOVA test following by the Tukey post hoc test. (* p<0,01) compared to control data.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475694&req=5

pone-0047468-g009: Cytosolic and nuclear P-β-catenin expression in MB cell lines.Phospho-β-catenin expression was determined by (A) Immunofluorescence and (B) Western blotting assays as described in Materials and Methods. For immunofluorescence, images were captured at objective lens of 20×magnification and the cytosolic and nuclear Phospho-β-catenin immunoreactivity were observed at merge column. For Western blotting, relative Phospho-β-catenin levels of the cytosolic and nuclear fractions obtained by densimetric analysis of protein bands detected in Western blots were compared to GAPDH and HnRNPK expression levels, respectively. Bars represent mean ± SD of 3 independent experiments performed in triplicate. Data were compared by One-Way ANOVA test following by the Tukey post hoc test. (* p<0,01) compared to control data.
Mentions: β-catenin is a downstream effector of the Wnt canonical pathway and, as described before, it is important for MB tumor prognosis and staging [3], [5]. We used immunofluorescence and Western blotting assays for detecting phospho-β-catenin expression in the MB cell lines used here. Firstly, we observed that Daoy and ONS76 showed mainly nuclear immunoreactivity to phospho-β-catenin, while the D283 MB cell line revealed immunoreactivity for this protein in the cytosol (Figure 9A). These qualitative data were confirmed by Western blotting of cytosolic and nuclear fractions compared to their respective controls, GAPDH and HnRNPK (Figure 9B).

Bottom Line: On the other hand, D283 cell line did not hydrolyze AMP.It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity.In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas e da Saúde, UFRGS, Porto Alegre, Rio Grande do Sol, Brasil.

ABSTRACT
Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5'NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5'NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5'NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

Show MeSH
Related in: MedlinePlus