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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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Flow cytometry analysis of ecto-5′-NT/CD73 protein expression in Daoy, ONS76 and D283 MB cell lines.Confluent MB cell lines were trypsinized and prepared as described in Materials and Methods. After incubation with purified mouse anti-human CD73 antibody (1∶10) and labeling with Alexa fluor 555 Rabbit anti-mouse IgG (1∶100), cells were analyzed by flow cytometry. (A) Results are shown as the ratio of labeled over non-labeled cells. (B) Representative graphic of ecto-5′NT/CD73-positive cells. Statistical relevance was analyzed by the One-Way ANOVA test following by the Tukey post hoc test. The experiments were performed in triplicate. (**) p<0.001 compared to control data.
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pone-0047468-g008: Flow cytometry analysis of ecto-5′-NT/CD73 protein expression in Daoy, ONS76 and D283 MB cell lines.Confluent MB cell lines were trypsinized and prepared as described in Materials and Methods. After incubation with purified mouse anti-human CD73 antibody (1∶10) and labeling with Alexa fluor 555 Rabbit anti-mouse IgG (1∶100), cells were analyzed by flow cytometry. (A) Results are shown as the ratio of labeled over non-labeled cells. (B) Representative graphic of ecto-5′NT/CD73-positive cells. Statistical relevance was analyzed by the One-Way ANOVA test following by the Tukey post hoc test. The experiments were performed in triplicate. (**) p<0.001 compared to control data.

Mentions: To confirm the presence of ecto-5′NT/CD73 on the cell surface, we performed flow cytometry analysis as described in the Material and Methods section. Figure 8A confirms the data presented in the mRNA expression analysis, where the cell lines representative of primary MB (Daoy and ONS76) showed higher expression of ecto-5′NT/CD73 than the D283 MB cell line, representative of secondary MB. The results were further quantified and presented the following percentages of ecto-5′NT/CD73 membrane expression in each MB cell line: Daoy: 74.52% ±8.026, ONS76∶67.97±4.087 and D283∶3.115±1.393.


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)

Flow cytometry analysis of ecto-5′-NT/CD73 protein expression in Daoy, ONS76 and D283 MB cell lines.Confluent MB cell lines were trypsinized and prepared as described in Materials and Methods. After incubation with purified mouse anti-human CD73 antibody (1∶10) and labeling with Alexa fluor 555 Rabbit anti-mouse IgG (1∶100), cells were analyzed by flow cytometry. (A) Results are shown as the ratio of labeled over non-labeled cells. (B) Representative graphic of ecto-5′NT/CD73-positive cells. Statistical relevance was analyzed by the One-Way ANOVA test following by the Tukey post hoc test. The experiments were performed in triplicate. (**) p<0.001 compared to control data.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0047468-g008: Flow cytometry analysis of ecto-5′-NT/CD73 protein expression in Daoy, ONS76 and D283 MB cell lines.Confluent MB cell lines were trypsinized and prepared as described in Materials and Methods. After incubation with purified mouse anti-human CD73 antibody (1∶10) and labeling with Alexa fluor 555 Rabbit anti-mouse IgG (1∶100), cells were analyzed by flow cytometry. (A) Results are shown as the ratio of labeled over non-labeled cells. (B) Representative graphic of ecto-5′NT/CD73-positive cells. Statistical relevance was analyzed by the One-Way ANOVA test following by the Tukey post hoc test. The experiments were performed in triplicate. (**) p<0.001 compared to control data.
Mentions: To confirm the presence of ecto-5′NT/CD73 on the cell surface, we performed flow cytometry analysis as described in the Material and Methods section. Figure 8A confirms the data presented in the mRNA expression analysis, where the cell lines representative of primary MB (Daoy and ONS76) showed higher expression of ecto-5′NT/CD73 than the D283 MB cell line, representative of secondary MB. The results were further quantified and presented the following percentages of ecto-5′NT/CD73 membrane expression in each MB cell line: Daoy: 74.52% ±8.026, ONS76∶67.97±4.087 and D283∶3.115±1.393.

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