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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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Time kinetics of ecto-nucleotide pyrophosphatase/phosphodiesterase activity by MB cell lines.Following reaching confluence, Daoy (A), ONS76 (B) and D283 (C) MB cell were incubated with 0.5 mM 5′-TMP-p-Nph as described in Materials and Methods. The values represent mean ± SD from six independent experiments performed in triplicate. Specific activities were expressed as nmol ρ-nitrophenol/min/mg of protein.
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pone-0047468-g003: Time kinetics of ecto-nucleotide pyrophosphatase/phosphodiesterase activity by MB cell lines.Following reaching confluence, Daoy (A), ONS76 (B) and D283 (C) MB cell were incubated with 0.5 mM 5′-TMP-p-Nph as described in Materials and Methods. The values represent mean ± SD from six independent experiments performed in triplicate. Specific activities were expressed as nmol ρ-nitrophenol/min/mg of protein.

Mentions: Considering that E-NPPs can help to keep the ideal nucleotide levels in the extracellular medium [34] and that its expression was detected in the MB cell lines here analyzed, we evaluated the activity of these enzymes as described in Material and Methods. The three cell lines studied showed greater E-NPP activity in alkaline pH, the optimum pH for these enzymes in vitro[35]. However, at physiological pH, E-NPP enzymatic activity was extremely low in the MB cell lines, indicating its participation in the extracellular nucleotide metabolism even under these conditions (Figure 3).


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)

Time kinetics of ecto-nucleotide pyrophosphatase/phosphodiesterase activity by MB cell lines.Following reaching confluence, Daoy (A), ONS76 (B) and D283 (C) MB cell were incubated with 0.5 mM 5′-TMP-p-Nph as described in Materials and Methods. The values represent mean ± SD from six independent experiments performed in triplicate. Specific activities were expressed as nmol ρ-nitrophenol/min/mg of protein.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0047468-g003: Time kinetics of ecto-nucleotide pyrophosphatase/phosphodiesterase activity by MB cell lines.Following reaching confluence, Daoy (A), ONS76 (B) and D283 (C) MB cell were incubated with 0.5 mM 5′-TMP-p-Nph as described in Materials and Methods. The values represent mean ± SD from six independent experiments performed in triplicate. Specific activities were expressed as nmol ρ-nitrophenol/min/mg of protein.
Mentions: Considering that E-NPPs can help to keep the ideal nucleotide levels in the extracellular medium [34] and that its expression was detected in the MB cell lines here analyzed, we evaluated the activity of these enzymes as described in Material and Methods. The three cell lines studied showed greater E-NPP activity in alkaline pH, the optimum pH for these enzymes in vitro[35]. However, at physiological pH, E-NPP enzymatic activity was extremely low in the MB cell lines, indicating its participation in the extracellular nucleotide metabolism even under these conditions (Figure 3).

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