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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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Extracellular hydrolysis of phosphate esters by MB cell lines.After the confluence, the MB cells were incubated and the inorganic phosphate amount was analyzed as described in the Materials and Methods. (A) β-glycerophosphate (β-Gli-P), glucose-6-phosphate (Gli-6-P) and inorganic pyrophosphate (PPi), specific substrates to unspecific phosphatases were used to evaluate the enzymatic activity of these enzymes. (B) The MB cells were incubated with AMP in the presence of levamisole, a specific alkaline phosphatase inhibitor, to evaluate the influence of the phosphatases activity in AMP hydrolysis. Specific activities were expressed as nmol Pi/min/mg of protein. Data were compared by Two-Way ANOVA test following by Bonferroni post hoc test. All experiments were performed in triplicate.
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pone-0047468-g007: Extracellular hydrolysis of phosphate esters by MB cell lines.After the confluence, the MB cells were incubated and the inorganic phosphate amount was analyzed as described in the Materials and Methods. (A) β-glycerophosphate (β-Gli-P), glucose-6-phosphate (Gli-6-P) and inorganic pyrophosphate (PPi), specific substrates to unspecific phosphatases were used to evaluate the enzymatic activity of these enzymes. (B) The MB cells were incubated with AMP in the presence of levamisole, a specific alkaline phosphatase inhibitor, to evaluate the influence of the phosphatases activity in AMP hydrolysis. Specific activities were expressed as nmol Pi/min/mg of protein. Data were compared by Two-Way ANOVA test following by Bonferroni post hoc test. All experiments were performed in triplicate.

Mentions: Similar to AMP hydrolysis, the MB cell lines exhibited the same pattern of hydrolysis for other monophosphonucleosides (CMP, GMP, IMP and UMP) (Figure 6), as expected to ecto-5′NT/CD73 [17]. As it was shown before, Daoy and D283 expressed ALP mRNA, another enzyme that can hydrolyze AMP and other phosphate esters. Daoy and ONS76, which presented higher AMPase activity, revealed very low enzymatic activity in the presence of Glu-6P or β-Gly-P, two susbtrates for ALP (Figure 7A). D283 MB cells also poorly hydrolyzed these substrates. In addition, the potential participation of ALP activity in hydrolysis of AMP was evaluated by the presence of levamisole, a specific inhibitor of this enzyme (Figure 7B). AMPase activity in the MB cell lines was unchanged in the presence of the inhibitor, excluding ALP participation in AMP hydrolysis. We observed prominent PPi hydrolysis by all MB cell lines (Figure 7A). Inorganic pyrophosphatase activity was also detected in the three cell lines (Fig. 7A), being in agreement with the presence of an ecto-pyrophosphatase in all MB cell lines analyzed here.


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)

Extracellular hydrolysis of phosphate esters by MB cell lines.After the confluence, the MB cells were incubated and the inorganic phosphate amount was analyzed as described in the Materials and Methods. (A) β-glycerophosphate (β-Gli-P), glucose-6-phosphate (Gli-6-P) and inorganic pyrophosphate (PPi), specific substrates to unspecific phosphatases were used to evaluate the enzymatic activity of these enzymes. (B) The MB cells were incubated with AMP in the presence of levamisole, a specific alkaline phosphatase inhibitor, to evaluate the influence of the phosphatases activity in AMP hydrolysis. Specific activities were expressed as nmol Pi/min/mg of protein. Data were compared by Two-Way ANOVA test following by Bonferroni post hoc test. All experiments were performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0047468-g007: Extracellular hydrolysis of phosphate esters by MB cell lines.After the confluence, the MB cells were incubated and the inorganic phosphate amount was analyzed as described in the Materials and Methods. (A) β-glycerophosphate (β-Gli-P), glucose-6-phosphate (Gli-6-P) and inorganic pyrophosphate (PPi), specific substrates to unspecific phosphatases were used to evaluate the enzymatic activity of these enzymes. (B) The MB cells were incubated with AMP in the presence of levamisole, a specific alkaline phosphatase inhibitor, to evaluate the influence of the phosphatases activity in AMP hydrolysis. Specific activities were expressed as nmol Pi/min/mg of protein. Data were compared by Two-Way ANOVA test following by Bonferroni post hoc test. All experiments were performed in triplicate.
Mentions: Similar to AMP hydrolysis, the MB cell lines exhibited the same pattern of hydrolysis for other monophosphonucleosides (CMP, GMP, IMP and UMP) (Figure 6), as expected to ecto-5′NT/CD73 [17]. As it was shown before, Daoy and D283 expressed ALP mRNA, another enzyme that can hydrolyze AMP and other phosphate esters. Daoy and ONS76, which presented higher AMPase activity, revealed very low enzymatic activity in the presence of Glu-6P or β-Gly-P, two susbtrates for ALP (Figure 7A). D283 MB cells also poorly hydrolyzed these substrates. In addition, the potential participation of ALP activity in hydrolysis of AMP was evaluated by the presence of levamisole, a specific inhibitor of this enzyme (Figure 7B). AMPase activity in the MB cell lines was unchanged in the presence of the inhibitor, excluding ALP participation in AMP hydrolysis. We observed prominent PPi hydrolysis by all MB cell lines (Figure 7A). Inorganic pyrophosphatase activity was also detected in the three cell lines (Fig. 7A), being in agreement with the presence of an ecto-pyrophosphatase in all MB cell lines analyzed here.

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