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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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ATP, ADP and AMP hydrolysis by human MB cell lines.Confluent cultures of Daoy, ONS76 and D283 cells were incubated with ATP or ADP or AMP as described in the Materials and Methods section. For D283 cells, a concentration of 2.0 mM and an incubation time of 30 minutes were used for all substrates, whereas for Daoy and ONS76 cell lines, 1.0 mM and 30 minutes of incubation to ATP and ADP, and 2.0 mM and 10 minutes of incubation to AMP were used. Specific activities were expressed as nmol Pi/min/mg of protein. Bars represent mean ± SD of four independent experiments performed in triplicate. Data were compared by Two-Way ANOVA test following by Bonferroni post hoc test. (***) p<0.001 and (*) p<0.05 compared to control data.
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pone-0047468-g004: ATP, ADP and AMP hydrolysis by human MB cell lines.Confluent cultures of Daoy, ONS76 and D283 cells were incubated with ATP or ADP or AMP as described in the Materials and Methods section. For D283 cells, a concentration of 2.0 mM and an incubation time of 30 minutes were used for all substrates, whereas for Daoy and ONS76 cell lines, 1.0 mM and 30 minutes of incubation to ATP and ADP, and 2.0 mM and 10 minutes of incubation to AMP were used. Specific activities were expressed as nmol Pi/min/mg of protein. Bars represent mean ± SD of four independent experiments performed in triplicate. Data were compared by Two-Way ANOVA test following by Bonferroni post hoc test. (***) p<0.001 and (*) p<0.05 compared to control data.

Mentions: Next, we aimed to analyze E-NTPDase enzymatic activities. For this purpose, differential hydrolysis pattern of ATP, ADP and AMP by Daoy, ONS76 and D283 cell lines were investigated. All MB cell lines presented a low rate of hydrolysis of ATP and ADP (Figure 4). However, when we analyzed AMP hydrolysis, the enzymatic activities of the MB cell lines exhibited a specific and differential profile with Daoy > ONS76> D283. The specific activities were 74.11±8.33, 9.206±0.730 and 3.654±0.157 nmol Pi/min/mg protein, respectively, showing the highest AMPase activities in the Daoy cell line.


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)

ATP, ADP and AMP hydrolysis by human MB cell lines.Confluent cultures of Daoy, ONS76 and D283 cells were incubated with ATP or ADP or AMP as described in the Materials and Methods section. For D283 cells, a concentration of 2.0 mM and an incubation time of 30 minutes were used for all substrates, whereas for Daoy and ONS76 cell lines, 1.0 mM and 30 minutes of incubation to ATP and ADP, and 2.0 mM and 10 minutes of incubation to AMP were used. Specific activities were expressed as nmol Pi/min/mg of protein. Bars represent mean ± SD of four independent experiments performed in triplicate. Data were compared by Two-Way ANOVA test following by Bonferroni post hoc test. (***) p<0.001 and (*) p<0.05 compared to control data.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0047468-g004: ATP, ADP and AMP hydrolysis by human MB cell lines.Confluent cultures of Daoy, ONS76 and D283 cells were incubated with ATP or ADP or AMP as described in the Materials and Methods section. For D283 cells, a concentration of 2.0 mM and an incubation time of 30 minutes were used for all substrates, whereas for Daoy and ONS76 cell lines, 1.0 mM and 30 minutes of incubation to ATP and ADP, and 2.0 mM and 10 minutes of incubation to AMP were used. Specific activities were expressed as nmol Pi/min/mg of protein. Bars represent mean ± SD of four independent experiments performed in triplicate. Data were compared by Two-Way ANOVA test following by Bonferroni post hoc test. (***) p<0.001 and (*) p<0.05 compared to control data.
Mentions: Next, we aimed to analyze E-NTPDase enzymatic activities. For this purpose, differential hydrolysis pattern of ATP, ADP and AMP by Daoy, ONS76 and D283 cell lines were investigated. All MB cell lines presented a low rate of hydrolysis of ATP and ADP (Figure 4). However, when we analyzed AMP hydrolysis, the enzymatic activities of the MB cell lines exhibited a specific and differential profile with Daoy > ONS76> D283. The specific activities were 74.11±8.33, 9.206±0.730 and 3.654±0.157 nmol Pi/min/mg protein, respectively, showing the highest AMPase activities in the Daoy cell line.

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