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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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Related in: MedlinePlus

Metabolism of extracellular AMP by MB cell lines.Confluent cultures of Daoy (A), ONS76 (B) and D283 cells (C) were cultivated in 24 well plates until confluence and incubated with 100 µM AMP in 250 µL of incubation medium as described in Materials and Methods. Aliquots of the supernatant were collected at 0, 5, 10, 20, 30, 60 and 90 min, and levels of AMP, ADO, INO and HIPO (hipoxantine) were determined by HPLC. Data were expressed as mean ± SD values from two independent experiments performed in triplicate.
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pone-0047468-g005: Metabolism of extracellular AMP by MB cell lines.Confluent cultures of Daoy (A), ONS76 (B) and D283 cells (C) were cultivated in 24 well plates until confluence and incubated with 100 µM AMP in 250 µL of incubation medium as described in Materials and Methods. Aliquots of the supernatant were collected at 0, 5, 10, 20, 30, 60 and 90 min, and levels of AMP, ADO, INO and HIPO (hipoxantine) were determined by HPLC. Data were expressed as mean ± SD values from two independent experiments performed in triplicate.

Mentions: AMPase activity of MB cell lines was further studied by HPLC. Figure 5 shows that AMP was efficiently hydrolyzed by Daoy and ONS76 MB cell lines. These cells hydrolyze AMP almost entirely after 30 min of incubation; however, the profiles of hydrolysis were different. While Daoy converted AMP into ADO and accumulated this molecule at the end of the incubation time, ONS76 produced ADO, which probably was converted into INO; this molecule was accumulated as the main product of AMP metabolism. Confirming the results obtained by the colorimetric assay (Fig. 4), the D283 MB cell line did not hydrolyze AMP.


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)

Metabolism of extracellular AMP by MB cell lines.Confluent cultures of Daoy (A), ONS76 (B) and D283 cells (C) were cultivated in 24 well plates until confluence and incubated with 100 µM AMP in 250 µL of incubation medium as described in Materials and Methods. Aliquots of the supernatant were collected at 0, 5, 10, 20, 30, 60 and 90 min, and levels of AMP, ADO, INO and HIPO (hipoxantine) were determined by HPLC. Data were expressed as mean ± SD values from two independent experiments performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0047468-g005: Metabolism of extracellular AMP by MB cell lines.Confluent cultures of Daoy (A), ONS76 (B) and D283 cells (C) were cultivated in 24 well plates until confluence and incubated with 100 µM AMP in 250 µL of incubation medium as described in Materials and Methods. Aliquots of the supernatant were collected at 0, 5, 10, 20, 30, 60 and 90 min, and levels of AMP, ADO, INO and HIPO (hipoxantine) were determined by HPLC. Data were expressed as mean ± SD values from two independent experiments performed in triplicate.
Mentions: AMPase activity of MB cell lines was further studied by HPLC. Figure 5 shows that AMP was efficiently hydrolyzed by Daoy and ONS76 MB cell lines. These cells hydrolyze AMP almost entirely after 30 min of incubation; however, the profiles of hydrolysis were different. While Daoy converted AMP into ADO and accumulated this molecule at the end of the incubation time, ONS76 produced ADO, which probably was converted into INO; this molecule was accumulated as the main product of AMP metabolism. Confirming the results obtained by the colorimetric assay (Fig. 4), the D283 MB cell line did not hydrolyze AMP.

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